<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 5th August 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p>
<blockquote>
<h4>👉 <a href="https://lcms.labrulez.com/library"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></a></h4>
</blockquote>
<blockquote>
<p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p>
</blockquote>
<p>This week we bring to you applications, brochures and specifications from <strong>Agilent Technologies</strong>, <strong>Shimadzu</strong>, <strong>Metrohm</strong>, <strong>Thermo Fisher Scientific,</strong> and <strong>Waters Corporation</strong>!</p>
<h4><strong>1. Shimadzu: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/IVDR_White_Paper_2024_Shimadzu_737d95b5fb.pdf">How Shimadzu is simplifying your compliance workload for the new IVD regulation!</a></strong></h4>
<ul>
<li>Technical Note</li>
</ul>
<p><strong>What is the New EU In Vitro Diagnostic Regulation (IVDR)?</strong></p>
<p>New In Vitro Diagnostic Medical Device Regulation (IVDR) (EU) 2017/746 was published in EU Official Journal in May 2017 in order to ensure a high level of safety and health whilst supporting innovation. It applies from 26 May 2022 and replaced the previous IVD Directive (98/79/EC). This regulation set rules for in vitro diagnostic devices such as reagent, calibrator, kit, instrument, and software intended by the manufacturer to be used for diagnostic purposes.</p>
<p>IVDR retains some concepts of IVD Directive such as CE marking or essential requirements (under IVDR called “general safety and performance requirements”). As in the past, the manufacturers have to demonstrate that they and their devices meet the requirements through conformity assessment procedure.</p>
<p>Different from IVD Directive, however, IVDR introduced the riskbased classification (Figure 1). Under IVDR devices are classified in four: The devices with highest risks are classified in class D. The class A devices have low risks and include products for general laboratory use, accessories which possess no critical characteristics.</p>
<h4><strong>2. Shimadzu: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/LCMS_157_Determination_of_30_PFAS_Seafood_LCMS_8060_NX_060524_a44e3a68b5.pdf">Determination of 30 PFAS in Seafood by Liquid Chromatography Triple Quadrupole Mass Spectrometry (LC-MS/MS)</a></strong></h4>
<ul>
<li>Application</li>
</ul>
<p><strong>User Benefits</strong></p>
<ul>
<li>Validated method for 30 PFAS in seafood (shrimp) meeting all criteria of AOAC SMPR 2023.003</li>
<li>High precision, excellent recovery, low Limit of Quantification (LOQ)</li>
<li>Simple and rapid extraction using QuEChERS</li>
</ul>
<p><strong>Introduction:</strong> Per-and polyfluorinated substances (PFAS) are a diverse group of man-made chemicals used in numerous products since the 1950s. PFAS can enter the food supply by contact in environmentally contaminated areas, during food processing, or exposure to packaging. Because PFAS have been linked to serious health effects, accurate methodology is needed. In this application news, we describe a single laboratory validation study with a rapid extraction of low concentrations of 30 PFAS in shrimp using the QuEChERS technique followed by analysis using the Shimadzu Nexera Liquid Chromatograph coupled to a Shimadzu LCMS-8060NX triple quadrupole mass spectrometer (Figure 1). </p>
<p>We optimized the chromatography and instrument operating parameters to achieve excellent peak shape, separation, and sensitivity. Sensitivity was improved for 4  PFAS, PFOA, PFHxS, PFNA, and PFOS.</p>
<p>In this study, we spiked samples at three concentrations in triplicate. For greater accuracy, standards were matrixmatched and extracted and spikes were quantified using the isotope dilution technique. Recovery and precision were compared to the requirements of AOAC SMPR 2023.003. In addition, we determined the Limit of Quantitation (LOQ) as the lowest concentration meeting accuracy and precision, ion ratio, retention time, and signal-to-noise ratio criteria of the qualifier ion. All recovery, precision, and LOQ’s met the acceptance criteria of the SMPR. The target analytes, their acronym, chemical abstract number, and experimentally determined LOQ are shown in Table 1.</p>
<p><strong>Conclusion:</strong> The Shimadzu LCMS-8060NX Triple Quadrupole Mass Spectrometer coupled with a Shimadzu Nexera Liquid Chromatograph was used in a single laboratory study to measure 30 PFAS compounds in a shrimp matrix and compared to criteria set by AOAC SMPR 2023.003. Chromatography conditions and the mass spectrometer were optimized to achieve excellent separation of all analytes, baseline resolution between linear and branched isomers, and a two-minute separation between PFOS and potentially interfering cholic acids. </p>
<p>Precision and recovery and the experimentally determined LOQ are well within the requirements of the SMPR.</p>
<h4><strong>3. Agilent Technologies: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/technical_overview_lc_ms_intelligence_5994_7430en_agilent_b00bf96408.pdf">Boost Efficiency and Productivity with Instrument Intelligence and Workflow Automation</a></strong></h4>
<ul>
<li>Technical Note</li>
</ul>
<p><strong>Introduction:</strong> Ever-increasing pressure for laboratories to minimize operating costs while maximizing output demands innovative approaches to their daily work. Recent developments in artificial intelligence (AI) and automation can help to alleviate this growing burden. The goal is to get technology to contribute more and more to day-to-day operations by reducing errors and ensuring consistent high-quality data generation. For workflows using Agilent LC/TQ and LC/(Q-)TOF instruments, this goal is becoming a reality.</p>
<p>The latest generation of Agilent LC/TQ and LC/(Q-)TOF instruments offers a suite of intelligent automation features to keep the instruments at peak performance. SWARM autotune allows instruments to be tuned for specific applications and desired performance. Auto- and check tunes can be scheduled to run during downtime so that the instruments are always ready to go. Instrument status is actively monitored using Early Maintenance Feedback (EMF) with constant feedback so that users can anticipate and plan for maintenance. Data are actively examined to ensure high quality. And, if data-related issues are suspected, Intelligent Reflex reinjection workflows automatically work to correct the situation. The culmination of these instrument intelligence features reduces errors, rework, and manual intervention—boosting laboratory efficiency and productivity.</p>
<p><strong>Conclusion:</strong> Agilent LC/TQ and LC/(Q-)TOF instruments include numerous intelligence and workflow automation features to boost productivity and efficiency of demanding routine analysis applications. These intelligence features:</p>
<ul>
<li>Maximize instrument performance and analytical sensitivity (application-based SWARM autotune)</li>
<li>Minimize downtime (early maintenance feedback and scheduled tuning)</li>
<li>Maximize sample throughput (intelligent reflex and fast screening workflows) </li>
<li>Ensure that injected samples do not require repeat work(intelligent reflex carryover detection and above calibration range workflows)</li>
<li>Improve sample characterization (intelligent reflex targeted MS/MS confirmation and iterative MS/MS workflows)</li>
</ul>
<h4><strong>4. Agilent Technologies: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_peptide_impurity_two_dimensional_liquid_chromatograhy_5994_7654en_agilent_e71c92f265.pdf">Confirmation of Peptide-Related Impurity Intact Mass Using Agilent 1290 Infinity II Bio 2D-LC and InfinityLab LC/MSD XT</a></strong></h4>
<ul>
<li>Application</li>
</ul>
<p><strong>Abstract:</strong> This application note presents the analysis of peptide-related impurities for Semaglutide using an Agilent 1290 Infinity II bio 2D-LC and an Agilent InfinityLab LC/MSD XT system. Peptide-related impurities are challenging to separate from peptide-active pharmaceutical ingredients due to their structural diversity and relatively large molecular weights. To address this challenge, an Agilent AdvanceBio Peptide Plus column, which is stable under high-concentration acidic conditions, was used to achieve good separation of Semaglutide impurities. A solvent system compatible with mass spectrometry (MS) was used with the 1290 Infinity II bio 2D-LC and InfinityLab LC/MSD XT, enabling the acquisition of MS spectra. The deconvolution feature in Agilent OpenLab CDS software, version 2.8, facilitated the easy determination of molecular weights for impurity peaks analyzed by two-dimensional liquid chromatography (2D-LC).</p>
<p><strong>Conclusion:</strong> Generally, difficult-to-separate peptide-related impurities can benefit from improved resolution through optimizing the concentration of acidic modifiers and fine-tuning the column performance. However, confirming the molecular weight of impurities can be challenging due to mobile phases not being suitable for MS analysis. In this application note, a stability indicating test for Semaglutide demonstrated efficient transition to LC conditions compatible with MS analysis using the Agilent InfinityLab multiple heart-cutting valve feature of an Agilent 1290 Infinity II bio 2D-LC. An Agilent InfinityLab LC/MSD XT exhibited the sensitivity to confirm MS spectra, even when analyzing impurities at low concentrations in the second dimension, and the deconvolution feature of Agilent OpenLab CDS software, version 2.8, facilitated easy confirmation of accurate molecular weights. Based on these results, it is anticipated that this method will provide foundational data for the management of peptide-related impurities not only for formulation compatibility but also for stability studies.</p>
<h4><strong>5. Thermo Fisher Scientific: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/fl_002789_ccs_dnapac_rp_columns_fl002789_en_6747763b83.pdf">Revolutionize your oligonucleotide analysis with game-changing DNAPac RP columns</a></strong></h4>
<ul>
<li>Other</li>
</ul>
<p><strong>The semi-preparative format –  ideal for nucleic acid purification</strong></p>
<p>Product features</p>
<ul>
<li>Designed for ion-pair reversed-phase (IP-RP) LC and  LC/MS separations of both single-stranded oligonucleotides  and double-stranded nucleic acids</li>
<li>Unique selectivity of the DNAPac RP chemistry, now available  for puriﬁcation</li>
<li>Bioinert hardware to eliminate analyte adsorption and avoid  the need for column passivation</li>
<li>Wide operating pH range and high-temperature stability  (up to 110 °C)</li>
<li>High resolution and high throughput</li>
</ul>
<h4><strong>6. Thermo Fisher Scientific: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/fl_22070_dnapac_dnaswift_oligonucleotide_fl22070_en_99e4d8f460.pdf">Thermo Scientific DNAPac columns - Ultra-high resolution oligonucleotide separation</a></strong></h4>
<ul>
<li><p>Other</p>
</li>
<li><p><strong>Thermo Scientiﬁc™ DNAPac™ PA200 and DNAPac™ PA200RS Columns</strong> oﬀer the highest resolution charge based  oligonucleotide separations. These anion-exchange columns  provide unsurpassed resolution of full length from n–1, n+1 (and  other failure sequences). </p>
</li>
<li><p><strong>Thermo Scientiﬁc™ DNAPac™ RP (U)HPLC Columns</strong> are high-throughput, high-resolution columns designed for  ion-pair reversed-phase separations. They are compatible  with LC-MS, have long-lasting polymeric columns, and  can operate up to pH 14. They also have temperature  stability up to 110 °C. These columns can easily resolve  both small polynucleotides and large dsDNA fragments  up to 10,000 bp due to pores varying from 50-2000Å.  Now available in semi-preparative format.</p>
</li>
<li><p><strong>Thermo Scientiﬁc™ DNASwift™ SAX-1S Monolith Column</strong> provide high loading capacity for easy laboratory scale  charge-based puriﬁcation of oligonucleotide samples.</p>
</li>
</ul>
<h4><strong>7. Waters Corporation: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/fl_22070_dnapac_dnaswift_oligonucleotide_fl22070_en_99e4d8f460.pdf">VISUALIZATION AND QUANTIFICATION OF DRUG/METABOLITES BY SENSITIVE AND FAST TARGETED DESI IMAGING ON TQ SYSTEM</a></strong></h4>
<ul>
<li>Poster / <a href="https://lcms.labrulez.com/events/568"><strong>ASMS 2024</strong></a></li>
</ul>
<p><strong>INTRODUCTION:</strong> Mass spectrometry (MS) coupled to liquid chromatography (LC) has been typically used for drug metabolism and pharmacokinetics (DMPK) studies. However, tissues are homogenised and therefore the molecular localisation is lost.</p>
<p>Desorption electrospray ionization (DESI) mass  spectrometry imaging (MSI) has proven to successfully  map and independently quantify small molecules without  radioactive labelling. Including small pharmaceutical  molecules, as well as endogenous metabolites directly  from tissue without the need of sample preparation after  sectioning.</p>
<p>Tandem quadrupole (TQ) MS are renowned for their  sensitivity and specificity in targeted applications using  Multiple reaction monitoring (MRM) mode of acquisition.</p>
<p>Here, we used a commercial central nervous system  (CNS) active drug named pitolisant that treats  narcolepsy. It was dosed at pharmacological level and no  observed adverse effect level (NOAEL) to show the  distribution of the drug and its main metabolite in  different organs such as liver and brain using the  sensitive and fast DESI TQ system.</p>
<p><strong>CONCLUSION</strong></p>
<ul>
<li>Pitolisant and its metabolites were detected sub 0.1 ng  when spotted onto tissue section, demonstrating the  high sensitivity and specificity of targeted MRM MS  Imaging workflow.</li>
<li>A highly flexibility workflow is evidenced allowing high  speed DESI MSI experiments with no compromise in  sensitivity or including more MRM transitions to map  biomarkers as well as drug.</li>
<li>Pitolisant and its oxidized metabolite were easily  detected at pharmacological dose, illustrating the  different distributions in the liver and brain murine  tissue sections, information that wouldn’t be  discovered by traditional DMPK studies.</li>
<li>MSI quantification workflow is demonstrated with the  in-house MicroApp MSI Quantify.</li>
</ul>
<h4><strong>8. Waters Corporation: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/2024_asms_claude_q_tof_ae3ec65164.pdf">EVALUATION OF DESI MOUNTED ON Q-TOF AND TQ MASS SPECTROMETERS TO IMAGE PFAS COMPOUNDS IN DIFFERENT MATRICES</a></strong></h4>
<ul>
<li>Poster / <a href="https://lcms.labrulez.com/events/568"><strong>ASMS 2024</strong></a></li>
</ul>
<p><strong>INTRODUCTION:</strong> Per- and polyfluoroalkyl substances (PFAS) are a well-  known group of chemicals that have a variety of  commercial and consumer uses. They have been found  to be highly persistent and can accumulate in humans,  animals and the environment.</p>
<p>Traditionally, analysis of PFAS has focused on  determination with LC-MS/MS. Often laborious SPE  techniques are required before analysis of solid matrices,  such as food, plants or animal tissue to assess exposure  routes. Furthermore, samples are homogenized, and  therefore the information of where the PFAS accumulate  is not available.</p>
<p>Here we evaluate mass spectrometry imaging (MSI) to  detect PFAS directly from sample surfaces.  Figure 5. Multiple conformations of N-EtFOSAA and corresponding spe-  cific drift time ion images of the dilution series. Analyzed using SE-  LECT SERIES Cyclic IMS MS in IMS mode .  A desorption electrospray ionization (DESI) source  combined with two types of Q-Tof mass spectrometers  operating in full scan MS (with and without, and a high  performance tandem quadrupole mass spectrometer  operating in targeted multiple reaction monitoring (MRM)  mode were used to evaluate limits of detection (LODs) of  several PFAS compounds.</p>
<p><strong>CONCLUSION</strong></p>
<ul>
<li>61% and 55% of the PFAS compounds were detected as low as  10 ng/mL spotted on tissue when analyzed by negative mode  DESI in discovery full scan MS acquisition mode.</li>
<li>The targeted MRM acquisition mode on the DESI XS Xevo TQ  Absolute MS is the most sensitive system with 77% of the PFAS  compounds detected as low as 10 ng/mL spotted on tissue.</li>
<li>PFAS compounds have a different ion mobility, with faster  arrival time in the gas phase, allowing differential selection and  a decrease of interference signal.</li>
<li>Different conformations of several PFAS were detected by ion  mobility with only a single pass in the cyclic device.</li>
<li>Distinct distributions of PFAS in the seed and root of  contaminated PFAS lentils were demonstrated.</li>
</ul>
<h4><strong>9. Metrohm: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_eis_009_download_1f476a3249.pdf">Mott-Schottky Analysis - Using EIS for semiconductor applications</a></strong></h4>
<ul>
<li>Application</li>
</ul>
<p><strong>Using EIS for semiconductor applications:</strong> Semiconductors are intrinsic to modern life, but they stand to play an even greater role in the coming energy transition. Several techniques are employed in the pursuit of improved materials for energy production and storage. One particularly popular method for studying the electronic properties of potential new semiconducting materials is MottSchottky Analysis. The technique can be best understood as an extension of electrochemical impedance spectroscopy (EIS). By performing EIS at a range of different DC bias or offset potentials, the reciprocal of the square capacitance can be derived from the data and then plotted against the DC offset itself, producing a so-called Mott-Schottky Plot. From such a plot, critical parameters such as the carrier concentration, the doping profile, and the flatband potential can be conveniently extracted. This technique is popular across different fields, as it is versatile, non-destructive, and relatively fast. Compared to other options, Mott-Schottky Analysis also requires relatively little specialist equipment. This Application Note presents an example of MottSchottky measurement on a popular semiconducting material using VIONIC powered by INTELLO.</p>
<p><strong>CONCLUSIONS:</strong> In this Application Note, an example Mott-Schottky measurement is shown using VIONIC powered by INTELLO. The flatband potential and doping density have been calculated using NOVA and are comparable to values for this type of sample found in the literature.</p>


News from LabRulezLCMS Library - Week 32, 2024

<p><strong>In the research article published in ACS <em>Analytical Chemistry</em> journal the researchers from Van ’t Hoff Institute for Molecular Sciences (HIMS) of the University of Amsterdam, Queensland Alliance for Environmental Health Sciences (QAEHS) of The University of Queensland, and Agilent Technologies Deutschland GmbH presented a new algorithm for improving spectral cleanup in nontarget analysis.</strong></p>
<p>This study presents an algorithm for improving spectral cleanup in nontarget analysis (NTA) workflows by integrating both time and mass domain information. The algorithm uses a probability-based cumulative neutral loss (CNL) model to achieve efficient fragment deconvolution. When optimized, the model demonstrated a high true positive rate (TPr) of 95.0% and was tested on real samples, consistently achieving a TPr above 88.8% across various conditions. Compared to other methods, a combination of correlation analysis and the CNL model was most effective, providing robust results with an 84.7% TPr and significant reduction in spectral noise.</p>
<blockquote>
<h4><strong>The original article</strong></h4>
</blockquote>
<blockquote>
<h5><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c00896"><strong>Cumulative Neutral Loss Model for Fragment Deconvolution in Electrospray Ionization High-Resolution Mass Spectrometry Data</strong></a></h5>
<p>Denice van Herwerden, Jake W. O’Brien, Sascha Lege, Bob W. J. Pirok, Kevin V. Thomas, and Saer Samanipour</p>
<p><em>Analytical Chemistry</em> <strong>2023</strong> <em>95</em> (33), 12247-12255</p>
<p>DOI: 10.1021/acs.analchem.3c00896</p>
<h6>licensed under <a href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6>
</blockquote>
<h5>Selected sections from the article follow.</h5>
<h3><strong>Abstract</strong></h3>
<p>Clean high-resolution mass spectra (HRMS) are essential to a successful structural elucidation of an unknown feature during nontarget analysis (NTA) workflows. This is a crucial step, particularly for the spectra generated during data-independent acquisition or during direct infusion experiments. The most commonly available tools only take advantage of the time domain for spectral cleanup. Here, we present an algorithm that combines the time domain and mass domain information to perform spectral deconvolution. The algorithm employs a probability-based cumulative neutral loss (CNL) model for fragment deconvolution. The optimized model, with a mass tolerance of 0.005 Da and a scoreCNL threshold of 0.00, was able to achieve a true positive rate (TPr) of 95.0%, a false discovery rate (FDr) of 20.6%, and a reduction rate of 35.4%. Additionally, the CNL model was extensively tested on real samples containing predominantly pesticides at different concentration levels and with matrix effects. Overall, the model was able to obtain a TPr above 88.8% with FD rates between 33 and 79% and reduction rates between 9 and 45%. Finally, the CNL model was compared with the retention time difference method and peak shape correlation analysis, showing that a combination of correlation analysis and the CNL model was the most effective for fragment deconvolution, obtaining a TPr of 84.7%, an FDr of 54.4%, and a reduction rate of 51.0%.</p>
<h3><strong>Experimental Section</strong></h3>
<h4><strong>LC-ESI-Q-TOF Analysis</strong></h4>
<p>Chromatographic separation of analytes was performed using a <a href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC"><strong>1290 Infinity II LC system (Agilent Technologies, Germany)</strong></a>, consisting of a binary pump (G7120A), an autosampler (G7129B), and a column oven (G7116B). Samples were kept in the autosampler at room temperature (ca. 20 °C) and the injection volume was usually 1 μL. Analytes were separated on a <a href="https://lcms.labrulez.com/library?instrumentationItems=34&amp;mainauthorItems=10&amp;search=Poroshell+EC-C18"><strong>Poroshell EC-C18</strong></a> column (2.1 mm × 150 mm, 2.7 μm, Agilent Technologies) at a constant flow rate of 0.5 mL/min and a column temperature of 40 °C. The mobile phases were water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B). The following gradient program was used for the separation of analytes: at 0 min, 95% A; at 1 min, 95% A; at 21 min, 5% A; at 23 min, 5% A; at 23.1 min, 95% A; and at 28 min, 95% A. The HPLC system was connected to a <a href="https://lcms.labrulez.com/library?instrumentationItems=31-30-32-29&amp;mainauthorItems=10&amp;search=G6546A"><strong>G6546A quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies)</strong></a>, equipped with an electrospray ionization (ESI) source using the Dual Spray Agilent Jet Stream technology. The Q-TOF was operated in the high-resolution mode for the low (m/z 1700) mass range. The acquisition rate was set to 6 Hz performing all ion full scan measurements at alternating collision energies of 0, 20, and 40 eV. Data were always recorded for a mass range of m/z 50–1200 in profile storage mode. Ionization was performed in the positive mode and the ESI source was operated under the following conditions: a drying gas temperature of 225 °C, a drying gas flow of 12 L/min, a sheath gas temperature of 350 °C, a sheath gas flow of 11 L/min, and a nebulizer pressure of 35 psi. The capillary and nozzle voltages were kept at 3500 and 500 V, respectively. A reference solution, containing purine and hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine (HP-0921), was continuously supplied to the second sprayer of the ESI source using an isocratic pump (G7110B, Agilent Technologies, Germany).</p>
<h4><strong>Cumulative Neutral Loss Model</strong></h4>
<p>The CNL model was built based on the MassBank EU, MoNA, and NIST20 database entries that were obtained using electrospray ionization in positive mode with a mass resolution ≥5000, including any type of mass analyzer (i.e., Q-TOF and Orbitrap) and collision energy. (37−39) Where multiple entries for a single chemical were found, the spectra were merged to ensure that the CNLs for each compound would have equal contribution to the model, using a 0.001 Da mass window. It should be noted that 0.001 Da was not assumed to be the inherent uncertainty of the data and was used as bin width for generation of average spectra. To obtain the CNLs for each compound, the fragment m/z values from the merged spectra were subtracted from the precursor ion mass. This resulted in reducing the 360,750 individual spectra to 24,487 merged CNL spectra for unique chemical constituents. The CNLs were used in place of the fragments to better capture the structural information implicitly present in the spectra. For example, while a high-frequency fragment may not contain much structural information, a highly frequent CNL provides information on the parts of the structure that are detached during fragmentation. (40) For the CNL model building, a Bayesian (i.e., probabilistic) approach was employed to overcome the issues related to limited database and potential data leakage. (41)</p>
<p>These CNL spectra were used to build the true positive (TP) and true negative (TN) probability distributions that were required for the Bayesian CNL model. To calculate these, the CNL spectra were converted to binary vectors for a CNL range of 0–1000 Da with 0.001 Da steps (i.e., 1,000,001 CNL values). The TP binary vector, for each chemical, contains ones for CNLs found in the spectra and zeros for the remaining bits, while the TN binary vector contains ones for the CNLs that were not found in a spectrum (<a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c00896#fig1"><strong>Figure 1A</strong></a>). The CNL masses that were larger than the precursor ion were set to zero for the TN binary vector. The TP and TN CNL occurrence distributions were calculated by summing the binary vectors obtained for all 24,487 CNL spectra and adding 1 to each CNL bin to avoid obtaining probabilities equal to 0. Finally, the TP and TN probability distributions were obtained by dividing each CNL occurrence by the total number of TP and TN CNL occurrences, respectively. Overall, building the CNL model took about 1–2 h and depended on the number of spectra and unique chemicals in the databases.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2023_95_33_12247_12255_Figure_1_Workflow_figure_for_construction_of_the_CNL_model_A_and_an_example_calculation_for_using_the_CNL_model_with_a_dafcb42a5a.jpg" alt="Anal. Chem. 2023, 95, 33, 12247-12255: Figure 1. Workflow figure for construction of the CNL model (A) and an example calculation for using the CNL model with a mass tolerance of 0.005 Da and a scoreCNL threshold of 0.00 (B). The abbreviations are listed in (C)."/></p>
<p>These calculated TP and TN CNL probabilities were used as the conditional probability (i.e., P(B|A)) in the Bayes theorem (eq 1). Additionally, to be able to calculate the TP or TN probability of a precursor ion having a specific CNL (i.e., posterior probability or P(A|B)), a flat prior (P(A)) is assumed, and since the marginal probability (P(B)) is a constant normalizing factor, the Bayes theorem can be reduced to eq 2, meaning that the TP and TN probabilities given a certain CNL are proportional to the probability of a CNL given that it is a TP or TN.</p>
<p>(1) 𝑃(𝐴|𝐵)=𝑃(𝐵|𝐴)×𝑃(𝐴)𝑃(𝐵)</p>
<p>(2) 𝑃(𝐴|𝐵)∝𝑃(𝐵|𝐴)</p>
<p>Through eq 2, the TP and TN probabilities for a CNL can be obtained, which were used to calculate scoreCNL (eq 3). This score is used to evaluate whether a CNL belongs to a specific precursor ion mass. To calculate scoreCNL, the TP and TN sums of probability (i.e., ∑P(TP|CNL) and ∑P(TN|CNL), respectively) are obtained for specified CNL ± mass tolerance. For example, if the mass tolerance is set to 0.010 Da and a CNL mass of 18 has been found, then ∑P(TP) corresponds to the summed TP probabilities of the CNL masses of 17.99 to 18.01. After similarly calculating ∑P(TN), scoreCNL can be used to assess if the CNL in question relates to a true fragment mass of the precursor ion mass or not, corresponding to above or below the scoreCNL threshold, respectively. An example of calculating scoreCNL is depicted in Figure 1B.</p>
<p>(3) scoreCNL=1−∑𝑃(TN|CNL)/∑𝑃(TP|CNL)</p>
<h3><strong>Results and Discussion</strong></h3>
<h4><strong>Exploring the CNL Model</strong></h4>
<p>When building the CNL model, the TP and TN counts for each CNL were calculated. At first sight, a general trend of a decrease in the total number of CNL counts after a CNL of 125 Da was found (<a href="https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.3c00896/suppl_file/ac3c00896_si_001.pdf"><strong>Figure S1</strong></a>). This was expected, since the median of all precursor ions is ±300 Da and the higher CNLs are not possible (i.e., precursor ion mass &lt; CNLs) for more and more compounds. Additionally, a distribution of the fragment m/z counts was also generated for comparison (<a href="https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.3c00896/suppl_file/ac3c00896_si_001.pdf"><strong>Figure S2</strong></a>). Here, it can be seen that the fragment m/z counts are less clearly defined than the CNL distribution (<a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c00896#fig3"><strong>Figure 3</strong></a>). Finally, the CNL counts were converted to probabilities, and still, the same trend was observed (<a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c00896#fig3"><strong>Figure 3</strong></a>). However, the probabilities in the TN distribution are much smaller than the TP probability distribution due to the larger number of TN cases than TPs. Therefore, scoreCNL is calculated that evaluates the relationship between P(TP) and P(TN).</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2023_95_33_12247_12255_Figure_3_TP_and_TN_CNL_probability_distributions_that_are_implemented_in_the_CNL_model_A_Full_probability_distribution_b5806f45b0.jpg" alt="Anal. Chem. 2023, 95, 33, 12247-12255: Figure 3. TP and TN CNL probability distributions that are implemented in the CNL model. (A) Full probability distribution, (B) a zoomed-in fraction of the TP probability distribution, and (C, D) the TN probability distribution zoomed-in on the probability range and CNL range, respectively."/></p>
<p>To provide an idea of what these probabilities mean in terms of chemical information, an overview of a few CNL masses with high TP probability can be found in <a href="https://pubs.acs.org/doi/suppl/10.1021/acs.analchem.3c00896/suppl_file/ac3c00896_si_001.pdf"><strong>Table S2</strong></a> with the potential CHNO compositions of these CNL masses. The most prominent CNL has been found at a mass of 18.01, which corresponds to a commonly known neutral loss of water or H₂O. Other frequently occurring losses such as ammonia (NH₃), methanol (CH4O), and, when looking at larger CNLs, C₂H₄O₂ correspond to CNLs of 17.03, 32.03, and 60.02, respectively. These examples show that the CNL masses contain valuable information related to the neutral losses of the precursor ion, meaning that a fragment mass might not have the same m/z value for multiple precursor ions, while their CNL mass with that fragment is actually the same. Moreover, the CNL occurrence probabilities could potentially be used to assist fragmentation pattern prediction tools, by, for example, ranking the fragments based on their CNL occurrence probability.</p>
<h3><strong>Conclusions</strong></h3>
<p>In this paper, we showed a fragment deconvolution technique that is able to clean up LC-HRMS information, using only the information of the mass domain. For the measurements used to evaluate the performance, the CNL model, with the optimized parameters of a mass tolerance of 0.005 Da and a scoreCNL of 0.00, was able to maintain a TPr above 95%, and depending on the sample or aspect evaluated, FD rates between 33 and 79% and reduction rates between 9 and 45% have been found. Moreover, when time domain methods were combined with the CNL model, an optimal combination of correlation analysis with the CNL model was found, using a correlation threshold of 0.57 and the optimized CNL parameters. This combination was able to achieve a 51.0% reduction in the total number of fragments with a TPr of 84.7% and an FDr of 54.4%.</p>
<p>However, when evaluating the CNL range influence, the model performed best for the lower-range CNLs, which could be related to the higher number of TP and TN counts in this range. Therefore, it would be good if a larger number of spectra with an exact mass above 200 could be collected and included in the model. As the databases (i.e., MassBank EU, MoNA, and NIST) grow over time, the model can be easily rebuilt and optimized for the same data with the provided CNL model package. Additionally, the current model is built based on positive mode spectra. Since different fragments were found for the same chemical depending on the ionization mode, this paper focuses on the positive-mode CNL model due to the lower number of database spectra measured in negative mode. However, the developed CNLforFragments package could also be used to generate a negative-mode CNL model. Finally, when there are sufficient data available for all of the precursor ions, the potential to expand the model to obtain the likelihood of a CNL depending on the precursor ion mass could be investigated. However, there are currently too little data to implement this in the CNL model.</p>
<p>Overall, we showed the potential of a mass domain approach for the cleanup of fragments. The CNL model can be used when there is no time domain (e.g., for DDA) and assist existing methods when a time domain is present. Additionally, a score is calculated for each potential CNL mass, which could potentially be used as a prioritization technique to order the fragments based on true fragment likelihood. The developed algorithm is able to clean up MS2 spectra that can be fed to the structural elucidation workflows, ultimately resulting in highly confident identifications, independently from the workflow and the database. The incorporation of this model into the CompCreate.jl package for use with ULSA or other library search algorithms is an ongoing project in our group.</p>


Cumulative Neutral Loss Model for Fragment Deconvolution in Electrospray Ionization High-Resolution Mass Spectrometry Data

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 12th August 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p>
<blockquote>
<h4>👉 <a href="https://lcms.labrulez.com/library"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></a></h4>
</blockquote>
<blockquote>
<p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p>
</blockquote>
<p>This week we bring to you posters, applications, and other documents from <strong>Agilent Technologies</strong>, <strong>Shimadzu</strong>, <strong>Thermo Fisher Scientific,</strong> and <strong>Waters Corporation</strong>!</p>
<h4><strong>1. Agilent Technologies: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_amino_acid_ecd_6545xt_advancebio_5994_7506en_agilent_bbe30346d8.pdf">Identification of Amino Acid Isomers Using Electron Capture Dissociation in the Agilent 6545XT AdvanceBio LC/Q-TOF System</a></strong></h4>
<ul>
<li>Application</li>
</ul>
<p><strong>Abstract:</strong> Accurately determining the amino acid sequence is crucial for understanding a protein&#39;s structure and function. However, distinguishing between leucine (Leu) and isoleucine (Ile) poses a challenge, because they are positional isomers and cannot be differentiated using traditional collision-based fragmentation techniques. Electron‑based fragmentation offers a promising solution by producing side chain fragments that can be used to distinguish between Leu/Ile and other isobaric residues such as aspartate (Asp) and isoaspartate (isoAsp). This application note demonstrates the identification of isobaric residues in peptides and intact proteins using the Agilent 6545XT AdvanceBio LC/Q-TOF system equipped with an ExD cell for electron capture dissociation. Agilent ExDViewer software enables intuitive analysis of side chain fragmentation in Q-TOF datasets, enhancing the ability to discern between isobaric amino acids.</p>
<p><strong>Conclusion:</strong> This application note describes the analysis of isobaric amino acids using an Agilent 6545XT AdvanceBio Q-TOF LC/MS system equipped with an Agilent ExD cell and the Agilent ExDViewer software tool for analysis. Electron capture dissociation is a powerful fragmentation technique that provides complementary information to CID fragmentation. These methods can be applied to investigate peptide backbone and side chain fragmentation for a range of molecules, reducing ambiguity in protein sequence analysis. The sensitivity of the Q-TOF to detect ECD fragments combined with ExDViewer for fragment analysis provides an effective solution for targeted amino acid isobar identification.</p>
<h4><strong>2. Waters Corporation: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_amino_acid_ecd_6545xt_advancebio_5994_7506en_agilent_bbe30346d8.pdf">MODERNIZATION OF A LEGACY NORMAL PHASE METHOD ON A MODERN HPLC SYSTEM</a></strong></h4>
<ul>
<li>Poster / <a href="https://lcms.labrulez.com/companies/303"><strong>HPLC 2024</strong></a></li>
</ul>
<p><strong>INTRODUCTION</strong></p>
<p>In regulated laboratories, particularly those within the  pharmaceutical industry, High-Performance Liquid  Chromatography (HPLC) systems play a crucial role in  routine analysis. However, some challenges arise due to  legacy methods that were developed during the early  days of HPLC technology. These methods have not kept  pace with recent advancements and modernization in the  field and do not fully leverage the latest technological  improvements. Advancements in column chemistry and  system robustness allow for better separation and  detection of analytes.</p>
<p>Modern HPLC systems are more robust, reliable, and  capable of handling challenging analyses and can  accommodate various chromatographic modes, including  Normal Phase chromatography. While legacy methods  persist, regulatory agencies such as the United States  Pharmacopeia (USP) provide guidance on how to adapt  and adjust these methods. This helps bridge the gap  between legacy methods and modern HPLC systems.</p>
<p>In this study, we examine the modernization of a legacy  normal phase method, USP Propofol Assay, for use with a  HPLC system. The original method specified an older L3  column in a non-standardized column configuration (4.6  mm x 200 mm). Given these column specifications, we  compared a suitable column to coupling two columns to  match the specified column dimensions. We also  explored the use of a column that was scaled in  accordance with USP Chapter 6211 that meets the L/dp  requirement.</p>
<p><strong>CONCLUSION</strong></p>
<ul>
<li>Legacy columns, like the L3 used for column 1, has an  older packing technology and is unable to provide a  suitable chromatography. </li>
<li>The use of a newer packing as in BEH Columns helps provide a successful analysis</li>
<li>Coupling two columns, albeit successful, may prove to  be a much more expensive option and may be introducing more opportunities for issues to arise.</li>
<li>The third column derived from the use of the columns  calculator may be the more suitable route since it  adheres to the USP guidance as well as not being more  expensive and time consuming as the coupling of two columns.</li>
</ul>
<h4><strong>3. Waters Corporation: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/asms_2024_mccullagh_the_utility_of_cyclic_ion_mobility_to_improve_selectivity_and_analysis_efficiency_of_environmental_pfas_contamination_and_exposure_3f331eafb6.pdf">THE UTILITY OF CYCLIC ION MOBILITY TO IMPROVE SELECTIVITY AND ANALYSIS EFFICIENCY OF ENVIRONMENTAL PFAS CONTAMINATION AND EXPOSURE</a></strong></h4>
<ul>
<li>Poster / <a href="https://lcms.labrulez.com/companies/296"><strong>ASMS 2024</strong></a></li>
</ul>
<p><strong>INTRODUCTION</strong></p>
<p>Polyfluorinated alkyl substances (PFAS) exposure is a  potential contributor to increased cancer occurrence in the  human population. The bio-accumulative nature of PFAS  enables monitoring of levels in human biofluids to help gain  understanding into exposure levels and pathways. However  PFAS isomeric compounds, can be challenging to efficiently  separate using liquid chromatography.</p>
<p>Cyclic ion mobility (cIM) provides an added dimension of  separation and collision cross section (CCS) values can  provide a a complimentary identification descriptor. CCS  values provide an additional identification point across  multiple application areas. At low concentrations where  fragment information may be absent, CCS and arrival time  distribution (ATD) provide an additional identification point  alongside retention time (tr).</p>
<p>To highlight confidence of the utility of CCS values,  independent of ion mobility (IM) technology, when performing  PFAS non-targeted screening assays, we have used an  internally developed PFAS library to cross correlate PFAS  CCS reproducibility. The SELECT SERIESTM CyclicTM IMS  mass spectrometer (see Figure 1) has been used to perform  an inter-site/intra-site comparison and compared to published  drift tube (DT) ion mobility PFAS CCS values.1</p>
<p>Using LC-cIM-MS, branched and linear PFAS isomers have  been fully resolved. Detection of individual PFAS isomers  provides opportunity to correlate cancer risk with exposure to  specific PFAS isomers. Human serum samples were  analysed, and data generated for PFAS isomers and isobaric  biomarker isomer composition.</p>
<p>LC-cIM-MS (cIM resolution (R)<del>65-145) analyses were  performed using a SELECT SERIES Cyclic IMS mass  spectrometer. PFAS standards and human serum samples  were analysed, using a 22 min reversed phase separation  gradient. Using the combined peak capacity of LC-cIM,  enabled resolution of linear and branched isomers, whilst  simultaneously achieving an increase in analysis efficiency of  75 %. The enhanced resolution has the potential to facilitate a  time efficient correlation between PFAS isomeric structure,  concentration, and exposure. Linear and branched PFOS  isomers were IM resolved in anonymized human serum  extracts, using a 5.5-minute LC gradient. Importantly  chromatographic separation was achieved for PFOS and bile  acid biomarkers. Using ion mobility resolution (</del>145), we  report IM enhanced specificity and resolution for biomarker  isomers Taurodeoxycholic, Taurochenodeoxycholic and  Tauroursodeoxycholic acid.</p>
<p>A focus on LC-cIM-MS as a potential strategy to generate  time efficient, additional toxicological correlation specificity  with individual PFAS isomeric species and resultant elevation  of isobaric biomarker isomers is presented. Identification of  perfluoroalkyl carboxylic acids (PFCAs), cIM conformeric  profiles and corresponding CCS identification finger prints  provides newfound specificity.</p>
<h4><strong>4. Shimadzu: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/an_01_00757_en_5e7f73a1aa.pdf">Qualitative Analysis of Drug Metabolites Using LCMS-9050</a></strong></h4>
<ul>
<li>Application</li>
</ul>
<p><strong>User Benefits</strong></p>
<ul>
<li>The LCMS-9050 quadrupole time-of-flight (Q-TOF) mass spectrometer and LabSolutions Insight Explore™provide qualitative analysis of drug metabolites.</li>
<li>Analyze and Target Screening function in LabSolutions Insight Explore enables peak extraction of candidate drug metabolites</li>
</ul>
<p><strong>Introduction:</strong> After a drug is ingested into the body, it is metabolized by various reactions, including oxidation, reduction, hydrolysis, and conjugation. The prediction of drug metabolites is important because the metabolites may differ from the original drug in terms of efficacy and toxicity.</p>
<p>This Application News introduces an example of qualitative analysis of drug metabolites using the LCMS-9050 quadrupole time-of-flight (Q-TOF) mass spectrometer. Lenvatinib, a known anticancer drug, was administered orally to rats, and the metabolites in the liver were analyzed. Drug metabolite peaks were extracted using the Analyze function in LabSolutions Insight Explore. The Target Screening function narrowed down the candidate peaks based on a list of drug metabolites, and the MS and MS/MS spectra were analyzed in detail.</p>
<p><strong>Conclusion:</strong> Several metabolites derived from lenvatinib were detected by the LCMS-9050 quadrupole time-of-flight mass spectrometer and LabSolutions Insight Explore. By preparing a list of drug metabolites, it was possible to easily extract candidate metabolite peaks. This workflow can also be applied to the metabolite analysis of drugs otherthan lenvatinib.</p>
<h4><strong>5. Agilent Technologies: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/an_lib_electolyte_cary_3500_flexible_uv_vis_5994_7602en_agilent_ebebd9a4ad.pdf">Quality Control of Lithium-Ion Battery Electrolytes and Solvents by UV-Vis Spectroscopy</a></strong></h4>
<ul>
<li>Application</li>
</ul>
<h5><strong>Color measurements and safe chemical handling using an Agilent Cary 3500 Flexible UV-Vis with Cary Sipper pump</strong></h5>
<p><strong>Abstract:</strong> Ensuring the quality of electrolytes used in lithium-ion batteries (LIBs) is crucial for maintaining the safety, performance, and longevity of these energy storage devices. LIB electrolytes consist of lithium salts dissolved in organic solvents. Any discoloration (yellowness) of these near-clear solutions can indicate contamination or degradation. Industry methods such as ASTM D5386-16 Standard Test Method for Color of Liquids Using Tristimulus Colorimetry can be applied to assess the quality of electrolytes by conducting instrumental color measurements using UV-Vis spectroscopy. Given the low absorbance of colorless-to-near-colorless liquid samples like LIB electrolytes and solvents, a highly accurate and sensitive UV-Vis spectrophotometer is essential.</p>
<p>This study demonstrates an effective quality assessment method for LIB electrolytes and solvents using the Agilent Cary 3500 Flexible UV-Vis spectrophotometer fitted with the optional Agilent Cary Sipper flow cell pump. The method is suitable for production quality control of hazardous samples, ensuring safe sample handling, high throughput, and high quality results for production-critical chemicals.</p>
<p><strong>Conclusion:</strong> The Agilent Cary 3500 Flexible UV-Vis spectrophotometer provided accurate and precise color measurements for colorless to near-colorless LIB electrolytes and solvents in accordance with the ASTM D5386 standard method. Advantages of the Cary 3500 Flexible method and findings based on the data set include:</p>
<ul>
<li>Hazardous samples were introduced directly into a 10 mm path length flow cell using the Cary Sipper pump, ensuring user safety and high sample throughput without compromising data quality.</li>
<li>The superior sensitivity (high signal-to-noise ratio) of the Cary 3500 combined with Agilent Cary WinUV Color application software enabled the reliable monitoring and control of electrolyte properties by analyzing light absorption in the visible spectrum.</li>
<li>Higher Pt-Co color (APHA) values of the used LIB electrolytes compared to fresh samples suggested a deterioration in quality due to the color changes.</li>
<li>Color measurements could be used to improve the quality control of LIB electrolytes and solvents by identifying discoloration—an indication of contamination or degradation that could affect battery performance.</li>
<li>This effective method could be implemented in both manufacturing QC settings and R&amp;D laboratories.</li>
</ul>
<p>QC color measurements using the Cary 3500 Flexible UV-Vis would ensure that only high-quality electrolytes are used in the production of LIBs, enhancing their safety, efficiency, and durability.</p>
<h4><strong>6. Thermo Fisher Scientific: <a href="https://lcms.cz/labrulez-bucket-strapi-h3hsga3/xx_002781_lc_vanquish_method_moderization_guide_tip_sheet_xx002781_en_aca7492d0b.pdf">A Helpful Guide To Modernizing Your Old HPLC Methods</a></strong></h4>
<ul>
<li>Guide</li>
</ul>
<p><strong>Modernizing older or compendial methods can help increase your lab’s productivity and lower your cost and carbon footprint. This guide gives you an overview of what method parameters to consider changing, as defined by USP 621.</strong></p>
<h4><strong>7. Thermo Fisher Scientific: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/wp_003007_lsms_Stellar_LCMS_translational_research_ffe410ad04.pdf">Transitioning biomarkers from discovery to validation at unprecedented scale with the Stellar mass spectrometer</a></strong></h4>
<ul>
<li>Technical note</li>
</ul>
<h5><strong>Expanded target capacity, higher sensitivity with greater specificity with unrivaled quantitative productivity for translational research</strong></h5>
<p><strong>Keywords:</strong> Stellar mass spectrometer, biomarker verification, targeted quantitative solutions, protein, metabolite, lipid, dynamic retention time adjustment, automated method creation, fast polarity switching, translational research.</p>
<p><strong>Introduction:</strong> The Thermo Scientific™ Stellar™ mass spectrometer establishes a new paradigm to drive biomarker verification by achieving 10X the sensitivity for 5X more compounds at scale compared to existing technologies. By synergistically combining the robust quantitative performance of triple quadrupole technology with the sensitive, rapid full scan MSn acquisition of dual-pressure linear ion trap technology, the Stellar mass spectrometer extends the unprecedented analytical capabilities to a wider range of compounds. Single-ion detection capabilities expand robust targeted quantitation for single cell or low protein load studies minimizing the potential for missing data. Novel software tools streamline highly multiplexed targeted quantitative method creation, implementation, and data acquisition, bypassing lengthy and costly replicate injections associated with existing technology. The new experimental capabilities of the Stellar mass spectrometer make it ideally suited for transitioning putative biomarker candidates from discovery to validation for translational proteomics, metabolomics, and lipidomics research.</p>


News from LabRulezLCMS Library - Week 33, 2024

<p><strong>In the week of August 19, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.</strong></p>
<blockquote>
<h4><a href="https://lcms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></h4>
</blockquote>
<blockquote>
<p><strong>👉 Search and filter in the largest global database</strong>  with over <strong>3 500 GC, LC, MS and spectroscopy webinars</strong>.</p>
</blockquote>
<h5><strong>1. Agilent Technologies: Elevate Your Protein Characterization: Discover Agilent ExD</strong></h5>
<ul>
<li>Tu, 20.8.2024 06:30 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4198">Registration</a></li>
</ul>
<p>Learn how you can meet these challenges with the latest enhancements for peptide &amp; protein characterization with the Agilent 6545XT AdvanceBio LC/Q-TOF.</p>
<h5><strong>2. SCIEX: SCIEX OS software summer school - Data processing: quantitation</strong></h5>
<ul>
<li>Tu, 20.8.2024 11:00 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4157">Registration</a></li>
</ul>
<p>Join us for our summer school series dedicated to enhancing your proficiency with SCIEX OS software.</p>
<h5><strong>3. Agilent Technologies: Reaching Ultra-Trace PFAS Detection Limits by Overcoming Solvent Effects at High Injection Volumes</strong></h5>
<ul>
<li>We, 21.8.2024 17:00 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4165">Registration</a></li>
</ul>
<p>High concentration factor in sample preparation with the use of Feed Injection for improved chromatographic performance at high injection volumes and with LC/MS allows lowest possible PFAS detection limits.</p>
<h5><strong>4. Thermo Fisher Scientific: Single-cell spatial metabolomics and lipidomics using AP-SMALDI and Orbitrap technology</strong></h5>
<ul>
<li>Th, 22.8.2024 11:00 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4188">Registration</a></li>
</ul>
<p>Technical aspects of mass spectrometry imaging, including the use of a specialized objective lens and the importance of high lateral resolution and mass resolving power.</p>


Webinars LabRulezLCMS Week 34/2024

<p><strong>In the research article published in ACS <em>Analytical Chemistry</em> journal the international team of researchers from Vrije Universiteit Brussel, University of Salzburg, University of Natural Resources and Life Sciences Vienna, CAP Chromatography Consulting, California, and University of Salzburg presented a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography.</strong></p>
<p>This study delves into the mechanisms behind retention and selectivity in hydrophobic interaction chromatography (HIC) for profiling molecular variants of trastuzumab. By evaluating three column chemistries—butyl, alkylamide, and multialkylamide—it highlights the impact of column hydrophobicity, surface area, and the role of chloride ions in enhancing chromatographic selectivity. The multialkylamide column demonstrated improved selectivity due to protein intercalation and steric effects. Furthermore, <a href="https://lcms.labrulez.com/library?instrumentationItems=32"><strong>LC–MS/MS</strong></a> peptide mapping revealed that chloride in the mobile phase facilitated site-specific deamidation and oxidation, offering insights into structural changes impacting trastuzumab&#39;s retention behavior.</p>
<blockquote>
<h4><strong>The original article</strong></h4>
</blockquote>
<blockquote>
<h5><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c04011"><strong>Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants</strong></a></h5>
<p>Raphael Ewonde Ewonde, Katharina Böttinger, Jelle De Vos, Nico Lingg, Alois Jungbauer, Christopher A. Pohl, Christian G. Huber, Gert Desmet, and Sebastiaan Eeltink</p>
<p><em>Analytical Chemistry</em> <strong>2024</strong> <em>96</em> (3), 1121-1128</p>
<p>DOI: 10.1021/acs.analchem.3c04011</p>
<h6>licensed under <a href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6>
</blockquote>
<h5>Selected sections from the article follow.</h5>
<h3><strong>Abstract</strong></h3>
<p>This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography–mass spectrometry (LC–MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M₂₅₅, W₄₂₀, and M₄₃₁) was observed for peptides situated at the Fc region close to the CH2–CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.</p>
<h3><strong>Experimental Section</strong></h3>
<h4><strong>HIC Instrumentation and Conditions</strong></h4>
<p>HIC experiments were conducted using an <a href="https://lcms.labrulez.com/library?manufacturerItems=2&amp;search=Ultimate+3000+bioinert+HPLC"><strong>Ultimate 3000 bioinert HPLC</strong></a> system (Thermo Fisher Scientific, Germering, Germany) composed of a low-pressure gradient pump, a well-plate autosampler enabling inline split-loop injections, a forced-air column oven, and a diode-array UV detector. <a href="https://lcms.labrulez.com/library?manufacturerItems=2&amp;search=Chromeleon"><strong>Chromeleon</strong></a> software (version 7.2.10) was used for system control and data management. The injection volume was set to 5 μL. The temperature of the well-plate sampler was set at 6 °C and the column oven was maintained at 30 °C. A 2.5 μL UV flow cell was used at 210 and 280 nm with a data collection rate of 5 Hz and a response time of 1 s. The columns used in this study were 4.6 mm I.D. × 100 mm packed with 5 μm porous (1000 Å) silica particles functionalized with alkylamide chemistries (MabPac HIC-10 and MabPac HIC-20) or packed with 5 μm nonporous polymer particles functionalized with butyl chemistry (HIC-butyl) obtained from Thermo Fisher Scientific (Sunnyvale). HIC analyses were generally performed in gradient mode by applying inverse salt gradients starting at 2 M (NH4)2SO4 or 4 M NaCl in 50 mM phosphate buffer and decreasing the salt concentration linearly in time. All mobile phases were filtered through a 0.22 μm filter (Millipore, Darmstadt, Germany).</p>
<h4><strong>RP-HPLC-MS/MS Peptide Profiling</strong></h4>
<p>HPLC-MS/MS experiments were performed using an <a href="https://lcms.labrulez.com/library?manufacturerItems=2&amp;search=Ultimate+3000+RSLCnano"><strong>Ultimate 3000 RSLCnano</strong></a> coupled via nano-ESI (NSI) to a <a href="https://lcms.labrulez.com/library?manufacturerItems=2&amp;search=Q+Exactive+Hybrid+Quadrupole-Orbitrap"><strong>Q Exactive Hybrid Quadrupole-Orbitrap</strong></a> mass spectrometer (Thermo Fisher Scientific). Proteins were denatured with 5 mM tris(2-carboxyethyl)phosphine (Sigma-Aldrich) at 60 °C and 900 rpm for 30 min. Disulfides were alkylated using 20 mM iodoacetamide (Sigma-Aldrich) at 22 °C and 750 rpm in darkness for 45 min. Protein subunits were purified using C18 pipet tips (Thermo Fisher Scientific), dried under vacuum (ScanVac, Lynge, Denmark), and digested with trypsin (Promega, enzyme/protein ratio = 1:25) overnight at 37 °C.</p>
<p>Resulting peptides were separated using a 75 μm I.D. × 150 mm <a href="https://lcms.labrulez.com/library?manufacturerItems=2&amp;search=PepMap+Neo"><strong>PepMap Neo</strong></a> column packed with 2 μm C18 particles with 100 Å pores (Thermo Fisher Scientific) with 0.1% FA in H2O (A) and ACN (B) applying the following gradients: 1% B for 5 min, 1–30% B in 40 min, 30–60% B in 10 min, 99% B for 5 min, and 1% B for 20 min at 0.3 μL/min and 50 °C. The spray voltage for the NSI source probe was set at +1.4 kV, a probe heater temperature and capillary temperature at 350 and 250 °C, respectively, and an S-lens RF level at 60. Peptides were analyzed in a data-dependent MS/MS mode. Full scan was operated within a scan range of m/z 400–3000 at a resolution setting of 70,000, with an AGC target of 3e6, and a maximum injection time of 150 ms. Ten data-dependent scans were acquired at a normalized collision energy of 28 with a resolution setting of 17,500, an AGC target of 1e5, a maximum injection time of 150 ms, and a dynamic exclusion of 10 s. MS data were analyzed using Byonic (v. 3.11.3, Protein Metrics Inc., Cupertino). Peptides were quantified using <a href="https://lcms.labrulez.com/library?search=Skyline"><strong>Skyline</strong></a> (v. 20.2).</p>
<h3><strong>Results and Discussion</strong></h3>
<h4><strong>Retention and Selectivity Assessment</strong></h4>
<p>The retention characteristics of a mAb was evaluated on three HIC column chemistries, i.e., a conventional HIC phase based on butyl ligands (HIC-butyl), a bonded phase based on short strands of polymeric alkylamide, and a bonded phase based on long strands of a multialkylamide ligand. <a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c04011#tbl1"><strong>Table 1</strong></a> provides an overview of stationary phase properties.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2024_96_3_1121_1128_Table_1_Stationary_Phase_Characteristics_6522dcbf48.jpg" alt="Anal. Chem. 2024, 96, 3, 1121-1128 - Table 1. Stationary Phase Characteristics."/></p>
<p><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c04011#fig1"><strong>Figure 1</strong></a> shows the peak profiles of the trastuzumab reference sample injected on the different columns by applying ammonium sulfate (<a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c04011#fig1"><strong>Figure 1A</strong></a>) and sodium chloride (<a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c04011#fig1"><strong>Figure 1B</strong></a>) salt systems...</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2024_96_3_1121_1128_Figure_1_Comparison_of_retention_and_selectivity_obtained_on_different_HIC_columns_for_unstressed_trastuzumab_applying_inverse_d12d87b2f4.jpg" alt="Anal. Chem. 2024, 96, 3, 1121-1128: Figure 1. Comparison of retention and selectivity obtained on different HIC columns for unstressed trastuzumab, applying inverse gradients of NH₄(SO₄)₂ from 2 to 0 M (A) and NaCl from 4 to 0 M (B) on a butyl phase (black profile), a bonded phase based on short strands of polymeric amide (green profile), and a covalently attached stationary phase based on a multiamide ligand (red profile). F = 1 mL/min, tG = 10 min, and the column oven was maintained at 30 °C."/></p>
<h4><strong>Optimizing the HIC Resolving Power</strong></h4>
<p>To generate an IgG sample with a substantial amount of oxidized amino acid residues, forced oxidation with hydrogen peroxide was pursued. To optimize the resolution between the resulting antibody variants, the effects of gradient span (Δc), gradient duration (tG), and column length were systematically assessed by injecting the reference standard before and after oxidation. As the determination of the peak width (even at half height) did not result in reliable performance metric, the valley-to-peak ratio (V/P) introduced by Christophe was used to evaluate performance, where V is the height of the valley between the critical peak pair and P is the height of the lowest peak, the lower the V/P, the better the separation. (26) <a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c04011#fig3"><strong>Figure 3A</strong></a> shows the separation of the reference sample (black trace) and the separation of the antibody variant generated after forced oxidation is shown in Figure 3B (red trace) obtained on a 100 mm long column after optimizing Δc (3–0 M) and tG (30 min). When applying only a narrow gradient span from 2 to 0 M, the critical pair elutes close to the column hold-up time and the gradient duration is only utilized to a very small extent, resulting in an increase in V/P and decreasing the resolving power (data not shown). In the next step, the flow rate and gradient duration were systematically varied. Applying Δc of 3–0 M, a flow rate of 1 mL/min in combination with a tG of 30 min yielded a V/P of 0.40. Increasing tG led to a slight decrease in resolving power as the peak width increased linearly with gradient duration. On the other hand, reducing the flow rate to 0.5 mL/min led to an improved separation, yielding a V/P of 0.34. Operating at a lower flow rate results in a decrease in the mass-transfer contribution to the overall peak broadening, surpassing the impact of gradient steepness (tG/t0) on resolving power. To further optimize the separation, the effect of column coupling was assessed. Applying two coupled columns led to a further decrease in V/P, yielding 0.31, see Figure 3C (reference sample) and Figure 3D (trastuzumab after forced oxidation), respectively. Note that tG/t0 was scaled to column length, allowing an unbiased comparison with the performance obtained on one column. While one column displays limited resolution, novel oxidation variants become apparent in the coupled-column experiment.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2024_96_3_1121_1128_Figure_3_Separation_of_the_trastuzumab_reference_sample_and_trastuzumab_variants_after_forced_oxidation_on_one_column_A_B_and_f3dc8402cd.jpg" alt="Anal. Chem. 2024, 96, 3, 1121-1128:Figure 3. Separation of the trastuzumab reference sample and trastuzumab variants after forced oxidation on one column (A, B) and two serially coupled columns (C, D). Separations were performed using the multialkylamide stationary phase, applying an inverse gradient of NaCl from 3 to 0 M, F = 0.5 mL/min, tG = 30 min (single column) and tG = 60 min (coupled columns). The fractions that were collected for subsequent RP-HPLC-MS/MS peptide mapping experiments, highlighted by the arrows (C, D)."/></p>
<h3><strong>Conclusions</strong></h3>
<p>We highlight the critical role of phase systems for tuning chromatographic retention and selectivity. To effectively profile molecular mAb variants, a stationary phase chemistry offering complementary interaction modes, such as van der Waals interactions, H-bonding, π–π interactions, and steric interactions, presents abundant opportunities for unparalleled selectivity. In addition, protein–salt interactions can play a key role in achieving high-resolution HIC separation. While (NH4)2SO4 is widely regarded as the gold standard for HIC, chloride ions have been identified as a crucial factor in achieving selectivity, leading to the resolution between distinct molecular antibody variants. Although the linear solvent strength (LSS) plots for mAbs are relatively steep; retention in HIC is not based on an on–off mechanism. Consequently, the increasing column length has a profoundly positive effect on the resolution. This also indicates that the chromatographic performance can likely be further optimized when downscaling the particle diameter and increasing the operating pressure while ensuring protein conformation is not affected. Nonetheless, selectivity mediated by the ligand architecture and salt type should be prioritized during mAb variant analysis. Finally, our study also demonstrated that the site-specific deamidation of trastuzumab lowers the retention time. However, deamidated species can only be separated from the nondeamidated fraction in the presence of chloride ions. Also, site-specific oxidation induces protein unfolding, which, in turn, increases the retention time due to increased hydrophobic residue exposure.</p>


Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants

<p><strong>In the week of August 26, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.</strong></p>
<blockquote>
<h4><a href="https://lcms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></h4>
</blockquote>
<blockquote>
<p><strong>👉 Search and filter in the largest global database</strong>  with over <strong>3 500 GC, LC, MS and spectroscopy webinars</strong>.</p>
</blockquote>
<h5><strong>1. US HUPO: Spotlight on Trainees: US HUPO 2024 Poster Presenters</strong></h5>
<ul>
<li>Tu, 27.8.2024 20:30 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4189">Registrace</a></li>
</ul>
<p>Missed out on visiting all the posters in Portland? Join us to hear more about the exciting research your fellow trainees are working on! Hosted by US HUPO&#39;s ECR.</p>
<h5><strong>2. Agilent Technologies: Effortless UV-VIS Analysis with Long Pathlength Measurements</strong></h5>
<ul>
<li>Tu, 27.8.2024 21:30 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/3912">Registrace</a></li>
</ul>
<p>The Cary 3500&#39;s flexible sampling options make this concentration analysis more effortless than ever before.</p>
<h5><strong>3. Waters Corporation: Fundamentals and biotherapeutics applications of Multi-Angle and Dynamic Light Scattering</strong></h5>
<ul>
<li>We, 28.8.2024 17:00 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4190">Registrace</a></li>
</ul>
<p>We will delve into the fundamentals and advantages of SEC-MALS and DLS for the biophysical characterization of monoclonal antibodies (mAbs), conjugated proteins, and adeno-associated viruses (AAVs).</p>
<h5><strong>4. Agilent Technologies: Panel Discussion: The Present &amp; Future of PFAS analysis – will we ever fully characterize PFAS?</strong></h5>
<ul>
<li>We, 28.8.2024 17:00 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4166">Registrace</a></li>
</ul>
<p>Panel Discussion: The Present &amp; Future of PFAS analysis – will we ever fully characterize PFAS?</p>
<h5><strong>5. Agilent Technologies: Getting the Most from Your Diode Array Detector: From Selection to Optimization</strong></h5>
<ul>
<li>Th, 29.8.2024 17:00 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/3913">Registrace</a></li>
</ul>
<p>We will cover the fundamentals of how your DAD works. From there we will discuss the various options for DAD method optimization.</p>
<h5><strong>6. US HUPO: Spatial Single-Cell Mass Spectrometry in Liver Tissue Slices: Experimental Design and Pipelines for Analysis</strong></h5>
<ul>
<li>Fr, 30.8.2024 17:00 CEST</li>
<li><a href="https://lcms.labrulez.com/webinar/4192">Registrace</a></li>
</ul>
<p>Join the MS Imaging SIG for an interactive discussion with authors on experimental design and data pipelines for analysis.</p>


Webinars LabRulezLCMS Week 35/2024

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 19th August 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p>
<blockquote>
<h4>👉 <a href="https://lcms.labrulez.com/library"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></a></h4>
</blockquote>
<blockquote>
<p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p>
</blockquote>
<p>This week we bring to you posters from <a href="https://gcms.labrulez.com/events/568"><strong>ASMS 2024</strong></a> by <strong>Agilent Technologies</strong>, <strong>Shimadzu</strong>, <strong>Thermo Fisher Scientific,</strong> and <strong>Waters Corporation</strong>!</p>
<h4><strong>1. Agilent Technologies: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_6475_spe_pfas_semiconductor_asms_2024_wp496_en_agilent_cf6e3f6528.pdf">Quantitative Analysis of Legacy &amp; Emerging PFAS in Semiconductor Lubricant using Triple Quadrupole LC/MS</a></strong></h4>
<ul>
<li>poster / <a href="https://gcms.labrulez.com/companies/296"><strong>ASMS 2024</strong></a></li>
</ul>
<p><strong>Introduction:</strong> Per- and polyfluoroalkyl substances (PFAS) are known to play an important role in the semiconductor industry. For example, PFAS-containing lubricants used in manufacturing processes mainly include perfluoropolyether (PFPE), polytetrafluoroethylene (PTFE) and others that are believed to be of very low toxicity.¹ With environmental regulations &amp; manufacturing restrictions continuing to expand in their scope, the semiconductor industry is facing the challenge of sustainability.² Reliable workflows are needed to ensure effective monitoring and control of PFAS in upstream processes. This study established a workflow based on solid phase extraction (SPE) and LC/MS/MS for analyzing over 100 PFAS from semiconductor lubricant matrix. </p>
<p><strong>Conclusions:</strong></p>
<ul>
<li>A reliable workflow for quantitation of trace levels of PFAS in semiconductor lubricant, based on SPE cleanup and LC/MS/MS was developed.</li>
<li>The Agilent Bond Elut PFAS WAX cartridge offered selective and effective extraction for matrix cleanup and preconcentration of PFAS analytes in lubricant.</li>
<li>The workflow performance confirmed the applicability of the Agilent PFAS MRM Database and eMethod, for quantitation of PFAS using the Agilent 6475 LC/TQ system.</li>
<li>This workflow enables a ready-to-use protocol for lubricant suppliers and semiconductor manufactures to monitor trace levels of critical PFAS in lubricants.</li>
</ul>
<h4><strong>2. Shimadzu: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/MP_220_Identification_of_Double_Bond_Position_in_Cyclic_Ester_using_OAD_TOF_system_5918fdc1f4.pdf">Identification of Double Bond Position in Cyclic Ester using OAD TOF system</a></strong></h4>
<ul>
<li>poster / <a href="https://gcms.labrulez.com/companies/296"><strong>ASMS 2024</strong></a></li>
</ul>
<h5><strong>Overview</strong></h5>
<p>Oxygen Attachment Dissociation (OAD) (x1 000 000) is a novel method for fragmentation providing additional fragmentation to CID on the LCMS 9050 To enhance identification of cyclic ester(lactone) which is one of the aromatic ingredients OAD-MS/MS has been applied to provide double bond specific dissociation enabling C=C position assignments.</p>
<p><strong>Introduction:</strong> Lactones Lactones, which are aromatic aromatic ingredients find wide applications in perfumes detergents, and foods The aroma is derived from a combination of various compounds rather than single compound The presence of different types and amounts of impurities in these compounds whether synthesized or purified from natural products can result in distinct aromas To accurately evaluate the aroma it is crucial to identify the precise structural information of the compound including impurities However when the compound has a stable cyclic structure such as lactone direct access to the double bond position within the ring by the neutral radical generated through OAD becomes challenging. In this study, various lactones were hydrolyzed to convert the cyclic ester into a hydroxycarboxylic acid, enabling the identification of the double bond position.</p>
<h5><strong>Conclusions:</strong></h5>
<ul>
<li>Hydrolyzation combined with OAD MS/MS was worked to identify C=C bond positions of cyclic ester.</li>
<li>It was suggested that there are several isomers of Lactone B other than the position of the C=C bond.</li>
<li>OAD-MS/MS can distinguish isomers that cannot be identified by CID.</li>
</ul>
<h4><strong>3. Waters Corporation: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/2024_asms_stevens_pahs_micro_plastic_bf33e7cfcd.pdf">ANALYSIS OF HIGH MASS POLYCYCLIC AROMATIC HYDROCARBONS (PAHS) EXTRACTED FROM MICROPLASTICS SPILLED IN THE MARINE ENVIRONMENT</a></strong></h4>
<ul>
<li>poster / <a href="https://gcms.labrulez.com/companies/296"><strong>ASMS 2024</strong></a></li>
</ul>
<p><strong>INTRODUCTION</strong></p>
<p>In 2021 the container ship M/V X-Press Pearl  caught fire and later sank off the coast of Sri  Lanka. Over 1600 tons of polyethylene pellets,  aka nurdles, were aboard the ship along with 25  tons of nitric acid which began to leak and is  suspected to be the cause of the fire that lead to  the sinking. To add to the witches brew of  ingredients in this fire were fertilizers and  explosives being transported and flame  retardants deployed to douse the fire. When  combined with abundant, naturally-occurring  compounds found in the marine environment,  the need arises to characterize the composition  of intact, burned, and broken pieces of the  spilled plastic later collected from kilometers of  Sri Lankan beaches¹.</p>
<p>Determining the distribution and abundance of  PAHs in the samples is important for  characterizing the source of the microplastics  (MPs) and in selecting appropriate handling,  disposal and/or recycling methods. Both  traditional and advanced GC/MS methods such  as 2D GC are suitable for the determination of  PAHs up to 302 Da which is useful for such  purposes as the differentiation of pyrogenic  versus petrogenic inputs2. However, the toxicity  characteristics of samples has demonstrated  poor correlation with low mass (&lt;302 Da) PAHs  in past studies3. For that reason this work was  aimed at investigating the routine analysis of  higher mass PAHs in the range of 314 to 424 Da  to provide access to additional analytes that  may better correlate with toxicity as well as to  provide the possibility of additional source  markers.</p>
<p><strong>CONCLUSIONS</strong></p>
<ul>
<li>Analysis of an expanded range of high  molecular weight PAHs in unfractionated  extracts of MPs was achieved using a  standard GC-APCI QqQ configuration combined  with  asimple,  generic  METHODS  microextraction</li>
<li>Charge transfer, atmospheric pressure  chemical ionization facilitated the  implementation of a class-specific MRM  acquisition scheme</li>
<li>Only minor modification to the GC method  used for lower mass PAHs is required</li>
<li>Nitrogen carrier gas achieved elution of  HWM PAHs with symmetric, narrow peaks  through the use of flow programming</li>
<li>Further investigation of HMW PAHs for  potential source markers and toxic  compounds is enabled with this approach</li>
</ul>
<h4><strong>4. Thermo Fisher Scientific: <a href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_carbon_nitrogen_isotopomer_orbitrap_stellar_ms_hybrid_po_en_0e1e253794.pdf">Carbon and Nitrogen Positional Isotopomer Determination in Metabolites using Orbitrap IQ-X and Stellar MS - a novel hybrid nominal mass instrument</a></strong></h4>
<ul>
<li>poster / <a href="https://gcms.labrulez.com/companies/296"><strong>ASMS 2024</strong></a></li>
</ul>
<h5><strong>Abstract</strong></h5>
<p><strong>Purpose:</strong> Development of a targeted MS²/MS³ approach for the determination of positional isotopomers.</p>
<p><strong>Methods:</strong> Initial data acquired on Thermo Scientific Orbitrap IQ-X Tribrid Mass Spectrometer was used for the structural annotation. A targeted MSn based assay utilizing the above data to get the structural information of the metabolites was developed on a novel hybrid nominal mass instrument.</p>
<p><strong>Results:</strong> A targeted MS²/MS³ method was established for the determination of positional isotopomers in Adenosine Monophosphate (AMP). The method was used for evaluation of the effect of hypoxia on cellular metabolism in an in-vitro model.</p>
<h5><strong>Conclusions</strong></h5>
<ul>
<li>A workflow for targeted stable isotope analysis of isotopomer is described.</li>
<li>The use of product scans with MSn and alternate fragmentation can give positional information of label using nominal mass resolution instrument.</li>
</ul>


News from LabRulezLCMS Library - Week 34, 2024

<p><strong>In the research article published in ACS <em>Analytical Chemistry</em> journal the researchers from the University of Geneva, Switzerland, and Waters Corporation took a deep dive into understanding the retention mechanism of oligonucleotides (ONs), evaluation of the applicability of the linear solvent strength (LSS) retention model, and exploration of the potential of ultra-short columns.</strong></p>
<p>Ion-pairing reversed-phase liquid chromatography (<a href="https://lcms.labrulez.com/webinars?search=IP-RP"><strong>IP-RPLC</strong></a>) serves as a key method for characterizing oligonucleotides and their impurities. This study delved into understanding the retention mechanisms of oligonucleotides, validating the linear solvent strength (LSS) retention model for molecules ranging from 3 to 30 kDa, and confirmed the model&#39;s ability to predict retention times accurately. It introduced the use of ultra-short 5 mm columns which facilitated rapid separations of oligonucleotide mixtures in just 30 seconds without compromising separation efficiency. Despite oligonucleotides displaying &quot;on-off&quot; elution patterns similar to smaller proteins, the effectiveness of these shorter columns suggests no further gains in selectivity or efficiency from using longer columns. This finding sets the stage for future research on more complex oligonucleotide formulations using this accelerated method.</p>
<blockquote>
<h4><strong>The original article</strong></h4>
</blockquote>
<blockquote>
<h5><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934"><strong>High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns</strong></a></h5>
<p>Honorine Lardeux, Szabolcs Fekete, Matthew Lauber, Valentina D’Atri, and Davy Guillarme</p>
<p><em>Analytical Chemistry</em> <strong>2023</strong> <em>95</em> (27), 10448-10456</p>
<p>DOI: 10.1021/acs.analchem.3c01934</p>
<h6>licensed under <a href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6>
</blockquote>
<h5>Selected sections from the article follow.</h5>
<h3><strong>Abstract</strong></h3>
<p>Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an “on–off” elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.</p>
<p>...</p>
<h3><strong>Experimental Section</strong></h3>
<h4><strong>Chemicals and Samples</strong></h4>
<p>Water was obtained from a Milli-Q water purification system from Millipore (Bedford, MA). LC-MS grade methanol (MeOH) was purchased from Thermo Fisher Scientific (Reinach, Switzerland). Oligonucleotides were purchased from Eurogentec (Seraing, Switzerland) and Integrated DNA Technologies (IDT, Leuven, Belgium). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, ≥99%), triethylamine (TEA, ≥99.5%), and RNase-free water were purchased from Sigma-Aldrich (Buchs, Switzerland).</p>
<h4><strong>Sample and Mobile Phase Preparation</strong></h4>
<p>100 μM oligonucleotide aliquots were initially prepared by reconstituting lyophilized material in the appropriate volume of RNase-free water and stored at −20 °C.</p>
<p>Equimolar oligonucleotide mixtures were prepared by mixing aliquots and diluting the products to 5 μM in RNase-free water prior to the IP-RPLC analysis (if not stated otherwise). Sample mixtures used to illustrate the retention of oligonucleotides were (first part of the study) the following: dT10–40 (a mixture of seven poly-deoxythymidylic (dT) acids of 10-, 15-, 20-, 25-, 30-, 35-, and 40-mer) and dT40–100 (a mixture of four poly-dT acids of 40-, 60-, 80-, and 100-mer). For the second part of the study, dT40–100, 4 dT (a mixture of four poly-dT acids of 10-, 20-, 30-, and 40-mer), and 4 PS (a mixture of four 20-mer poly-dT acids with 0, 6, 9, and 19 phosphorothioate modifications that replace the phosphodiester linkages) were used.</p>
<p>Mobile phase A was 14 mM TEA in water, containing 100, 160, or 400 mM HFIP, pH 8.2, 8.1, or 7.8, respectively. Mobile phase B was a mixture of 50:50 mobile phase A and methanol.</p>
<h4><strong>Chromatographic System, Columns, and Software</strong></h4>
<p>All chromatographic separations were performed on a Waters <a href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;manufacturerItems=5&amp;search=ACQUITY+UPLC+I-Class"><strong>ACQUITY UPLC I-Class</strong></a> System (Milford, MA) equipped with a binary solvent delivery pump, an autosampler with a flow-through-needle (FTN), and a UV detector with a 0.5 μL UV flow cell. The overall extra-column volume was measured as 5.6 μL from the injection seat of the autosampler to the detector cell, while the offset time was equal to 0.87 s. These values were found after plotting system residence time vs reciprocal flow rate (1/F) for various experiments conducted at several flow rates in the absence of a column (zero-dead volume union). Waters <a href="https://lcms.labrulez.com/library?from=2020&amp;mainauthorItems=5&amp;manufacturerItems=5&amp;to=2024&amp;search=ACQUITY+UPLC+BEH"><strong>ACQUITY UPLC BEH</strong></a> C18 1.7 μm Column, 5 mm × 2.1 mm, 130 Å <a href="https://lcms.labrulez.com/library?from=2020&amp;mainauthorItems=5&amp;manufacturerItems=5&amp;to=2024&amp;search=VanGuard+Pre-column"><strong>VanGuard Pre-column</strong></a> was used in this study. Commercial 50 mm and 150 mm × 2.1 mm columns (Waters ACQUITY Premier Oligonucleotide BEH C18 1.7 μm, 130 Å Column) were also used. Data acquisition and instrument control were performed by <a href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;manufacturerItems=5&amp;search=Empower"><strong>Empower</strong></a> 3 Software (Waters). The freely available Excel Spreadsheet developed by Guillarme et al. was used to derive LSS parameters and predict retention times. (23,24)</p>
<h4><strong>Apparatus and Methodology</strong></h4>
<p>Sample volumes of 1 μL were injected using linear gradients (for all separations). The column temperature was set at 60 °C unless stated otherwise. Various semi-empirical models are used in LC to describe the relationship between experimentally observed retention times and mobile phase composition or gradient conditions. In this study, the following model was considered...</p>
<h3><strong>Results and Discussion</strong></h3>
<h4><strong>Retention Properties of Oligonucleotides</strong></h4>
<p>Several studies have reported the elution behavior of ONs in IP-RPLC in the presence of various ion-pairing reagents and using different stationary phases. (25−27) Efforts were mainly focused on (1) improving the chromatographic resolution (selectivity and peak capacity) of ONs or (2) improving the LC-MS sensitivity. (25) Relative retention indices and retention time calibration of homo-oligonucleotides in IP-RPLC have been published, (28) but absolute dimensionless measures of retention (like the S parameter of the LSS model) have not been reported yet, at least to the best of our knowledge. In this study, we focus our attention especially on the sensitivity of solute retention to mobile phase composition (S parameter), as this determines the required (effective) column length. We also discuss the log k0 parameter that corresponds to the extrapolated value of k for φ = 0. Here, the change in mobile phase composition was approximated by the change of organic modifier composition (methanol), while neglecting the effect of the small change in additive concentration. (29) It has also been reported that IP-RPLC separations of ONs benefit from shallow gradients, as they elute in relatively sharp peaks, despite the shallow gradient applied. (25) This may indicate that the retention of the solute is very sensitive to the eluent composition (on–off-like behavior). In practice, 50–150 mm long columns are often used for the IP-RPLC analysis of ONs, which is not necessary if the on–off behavior is valid.</p>
<p>...</p>
<h4><strong>Illustration of the On–Off Mechanism of Oligonucleotides</strong></h4>
<p>In general, IP-RPLC acts as a mixed-mode chromatographic mode combining hydrophobic and electrostatic (ionic) retention mechanisms. However, if the ion-pairing reagent concentration is sufficiently high, elution is mainly driven by hydrophobic interactions occurring between the stationary phase ligands and the hydrophobic part (i.e., alkyl chain) of the ion-pairing reagent molecules.</p>
<p><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934#fig2"><strong>Figure 2</strong></a>A shows plots of the log retention factor as a function of mobile phase composition (φ) for ATP, dT20, dT100, and a monoclonal antibody (mAb) as a reference. The figure was constructed on the basis of data obtained with the 100 mM HFIP mobile phase, resulting in the highest S values. It clearly illustrates that dT20 already shows an on–off behavior (very steep curve, S = 60). For the dT100, we observed a 2.5 times higher S value (S = 264) compared with an intact mAb (S typically ranges between 100 and 150). The higher S values observed with ONs are probably due to the differences in molecular shape and specific surface available for interaction with the stationary phase (helical straight shape versus Y-shape).</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2023_95_27_10448_10456_Figure_2_Logarithmic_solute_retention_factor_d0e09e3597.jpg" alt="Analytical Chemistry 2023, 95 (27), 10448-10456 - Figure 2. Logarithmic solute retention factor (A) and relative migration velocity (u/u0) (B) as a function of the volume fraction of the organic mobile phase φ for ATP (black, MW = 0.5 kDa; S = 13), dT20 (green, MW = 6 kDa; S = 60), dT100 (red, MW = 30 kDa; S = 264), and an intact mAb (blue, MW = 150 kDa, S = ∼116)."/></p>
<p><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934#fig2"><strong>Figure 2</strong></a>B shows the relative migration velocity as a function of mobile phase composition. Such a plot illustrates the transition between fully adsorbed (“on”) and fully released (“off”) states. The value of S = 264 (and the log k0 = 45.2) means that up to a mobile phase composition of φ = 0.164, the migration velocity of the dT100 is practically zero (bound at the column inlet, its velocity is less than 1% of the interstitial mobile phase velocity), whereas from φ = 0.179, it moves at a velocity greater than 99% of the mobile phase velocity, indicating that the solute is fully desorbed from the column. The transition range between the adsorbed and desorbed states therefore corresponds to a Δφ range of only 1.5% (0.179–0.164). The transition range for the dT20 is somewhat wider, namely, 6.5% of Δφ. This transition typically occurs between 3 and 4% Δφ for an intact mAb in RPLC mode. For the small ATP molecule, we observed a transition range of 31.4% Δφ (from φ = 0.116–0.430).</p>
<p><a href="https://pubs.acs.org/doi/10.1021/acs.analchem.3c01934#fig3"><strong>Figure 3</strong></a> shows a plot of the column length (Leff) required to effectively retain dT20 and dT100 (at least at ke = 0.5, which is a common practice for small molecule separations) as a function of gradient time and flow rate.</p>
<p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Analytical_Chemistry_Anal_Chem_2023_95_27_10448_10456_Figure_3_Color_coded_surface_plot_of_the_column_length_Leff_required_to_retain_d_T20_and_d_T100_with_ke_0_5_7271adf747.jpg" alt="Analytical Chemistry 2023, 95 (27), 10448-10456 - Figure 3. Color-coded surface plot of the column length (Leff) required to retain dT20 and dT100 with ke = 0.5, as a function of gradient time and flow rate (dc = 2.1 mm ID, ε = 0.68, and Δφ = 0.2)."/></p>
<p>...</p>
<h3><strong>Conclusions</strong></h3>
<p>The goal of this proof-of-concept work was to evaluate if ultra-short columns can be used in IP-RPLC for the analysis of model oligonucleotides in gradient elution mode.</p>
<p>First, the validity of the LSS model was evaluated, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow the on–off elution behavior and that for most linear gradient separation conditions an effective column length between 5 and 35 mm was sufficient to obtain the best selectivity and separation efficiency. Therefore, the applicability of ultra-short columns was explored to speed up ON separations. In this regard, the extra-column band broadening induced by the instrumentation was carefully evaluated. Interestingly, the impact of injection volume and post-column connection tubing on peak capacity were found to be negligible. Next, the impact of column length on apparent efficiency was also monitored. First, column length was varied (150, 50, and 5 mm), while flow rate and gradient time were kept constant. Then, various gradient times and flow rates were tested on the three column lengths. In all cases, the on–off retention mechanism of ONs was confirmed. In addition, 5 mm column proved to be an advantageous choice to (i) increase the overall resolution achievable in gradient mode and (ii) perform ultra-fast separations with reasonable kinetic performance.</p>
<p>Optimized ultra-fast separations were therefore developed on the 5 mm column by using a flow rate of 1 mL/min and a gradient time of 1 min. Three model oligonucleotide mixtures including small, large, and PS-modified ONs were analyzed, and a remarkable resolution was especially obtained for the separation of the small ONs mixture. The separations were subsequently pushed to their limits at a higher flow rate (1.75 mL/min) and temperature (90 °C against the previous 60 °C), enabling baseline separation of all of the mixtures in as little as 30 s.</p>
<p>For the first time, a comprehensive study on the demonstration of the applicability of the LSS model to model oligonucleotides’ IP-RPLC separations in gradient elution mode was performed. The understanding of the ONs retention behavior enabled the evaluation of ultra-short columns and the selection of a 5 mm column to boost the ONs baseline separations in less than 1 min. These findings represent the first proof of concept for achieving ONs high-throughput analysis and pave the way for further investigation using more complex therapeutic oligonucleotides.</p>
<p>Interestingly, there are two possible reasons for using ultra-short columns for ONs: (i) achieving ultra-fast separation (1 min or less), while maintaining a reasonable efficiency, provided that the flow rate is sufficiently high and (ii) increasing the overall resolution achievable in gradient mode (corresponding to Pmin) within a reasonable analysis time (few minutes). The second aspect has not yet been explored but will be investigated in an upcoming work.</p>


High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 26th August 2024? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><h4>👉 <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></a></h4></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 to you posters from <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/events/568"><strong>ASMS 2024</strong></a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/events/592"><strong>52nd HPLC</strong></a> by <strong>Agilent Technologies</strong>, <strong>Shimadzu</strong>, <strong>Thermo Fisher Scientific,</strong> and <strong>Waters Corporation</strong>!</p><h4><strong>1. Agilent Technologies: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/enhancing_high_throughput_preparative_hplc_poster_06b9abadd0.pdf"><strong>Enhancing High Throughput Preparative HPLC: Offline Regeneration of Identical Superficially Porous Particle Preparative Columns Utilizing The 1290 Infinity II Preparative LC System and Agilent InfinityLab Poroshell 120 Preparative LC Columns to extend Column Lifetime, Minimize Sample Carryover and Increase Throughput</strong></a></h4><ul><li>poster / <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/companies/303"><strong>HPLC</strong></a></li></ul><p><strong>Abstact:</strong> High throughput sample purification and analysis are important tasks in many industries. With the increasing number of samples synthesized– HPLC purification should not be the bottleneck in discovery and development. The throughput on the Prep system can be increased by optimizing the chromatographic method and by optimizing the instrument hardware. In this poster we show how the throughput can be enhanced even further by using two identical Poroshell Preparative HPLC columns in the system and operating them alternately optimizing the 1290 Infinity II Preparative LC System for Off-Line Column Regeneration. The greatest benefit of the offline column cleanup and equilibration is the increase in sample throughput, but added benefits include extended column lifetime and a reduction in sample carryover.</p><p><strong>Conclusions:</strong> Faster run times and higher throughput is desirable when doing preparative HPLC. Using higher flow rates, shorter and lower pressure columns as well as optimized equipment can help to achieve these goals. Higher backpressure limits of the Agilent Inf II Prep pumps along with high pressure valves further reduce cycle times by allowing configuration of automated offline column regeneration. This means two columns are alternatively used via a 2-position/14-port valve. Cleaning and equilibration time can be excluded from the cycle time and 50% more runs were performed.</p><h4><strong>2. Waters Corporation: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/2024_hplc_maziarz_hydrophilic_hilic_f04a258724.pdf"><strong>Scaling Reverse-Phase and Hydrophilic Interaction Liquid Chromatography (HILIC) Methods to HPLC</strong></a></h4><ul><li>poster / <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/companies/303"><strong>HPLC</strong></a></li></ul><p><strong>INTRODUCTION:</strong></p><p>Achieving identical chromatographic separation and equivalent results are critical when scaling and migrating methods between different liquid chromatography (LC) instruments.</p><p>In this work, two different methods developed on 1.7 μm particle size columns using a low dispersion system were scaled to HPLC. In first example, a reverse-phase method for naphazoline hydrochloride, pheniramine maleate and associated related substances was scaled from 1.7 μm to a 2.5 μm particle column. A second method for the analysis of ribavirin and related compounds, operated under HILIC conditions, was scaled from 1.7 μm to 2.5 and 3.5 μm particle columns and run on different HPLC platforms. The scaled methods generated equivalent chromatographic performance and assay results as the original method.</p><p><strong>CONCLUSION:</strong></p><ul><li>Reverse-phase and HILIC methods were successfully scaled from 1.7 µm particle size columns to HPLC.</li><li>The target LC systems including ACQUITY Arc, Arc HPLC and Alliance iS HPLC Systems generated equivalent performance as the original method run on an ACQUITY UPLC H-Class Plus System.</li><li>Dwell and extra-column volumes were accounted for to ensure quality of the chromatographic data for method scaling and migration across different LC platforms.</li></ul><h4><strong>3. Thermo Fisher Scientific: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_003231_hplc_peptide_level_analysis_monoclonal_antibodies_evaluation_po003231_na_en_31f6f13cf1.pdf"><strong>Performance evaluation of a new reversed-phase column for peptide mapping and monitoring of monoclonal antibodies</strong></a></h4><ul><li>poster / <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/companies/303"><strong>HPLC</strong></a></li></ul><h5><strong>Abstract</strong></h5><p><strong>Purpose:</strong> Demonstrating the benefits of the newly introduced Thermo Scientific Hypersil GOLD Peptide C18 column with 1.9 µm particle and 175 Å average pore size, in the dimension of 150 x 2.1 mm (L x ID) for the analysis of tryptic mAb digest samples.</p><p><strong>Methods:</strong> UHPLC-MS/MS peptide mapping and UHPLC-UV-MS peptide monitoring.</p><p><strong>Results:</strong> The Hypersil GOLD Peptide column enables to uniquely meet the demands of peptide level analysis where consistent lot-to-lot and column-to-column performance is required.</p><h5><strong>Conclusions</strong></h5><p>The two studies of peptide mapping and peptide monitoring demonstrate the benefits of the Hypersil GOLD Peptide column for peptide level biopharma analysis based on a comprehensive dataset obtained from rituximab and adalimumab digest samples.</p><ul><li>SET and SST criteria of retention time and peak area precision are easily met when using the Hypersil GOLD Peptide column.</li><li>The Hypersil GOLD Peptide column provides<ul><li>peaks with good asymmetry values, strong retention time and peak area reproducibility.</li><li>retains and separates hydrophilic peptides and has very good chromatographic resolution values for deamidated species in the PENNYK region.</li><li>excellent column lot-to-lot reproducibility with % RSD RT ≤ 0.6, and % RSD peak area ≤ 6.2 for peptides across the entire elution window.</li><li>consistent column-to-column results for the quantification of glycopeptides with % RSD of relative abundance ≤ 3.0.</li></ul></li></ul><h4><strong>4. Shimadzu: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_2024_LEIPP_WP_119_45a784b1d5.pdf"><strong>Development of a blood-based multiplex quantitative LC-MS method for neurodegenerative diseases patient stratification : towards an innovative diagnostic tool</strong></a></h4><ul><li>poster / <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/companies/296"><strong>ASMS 2024</strong></a></li></ul><p><strong>Introduction and objectives:</strong> Current neurodegenerative biomarkers measurements for diagnosis purposes are performed using CSF samples which requires invasive procedures for the patient. Besides, AD and other associated dementias are very complex pathologies that can have intricate symptoms and pathological deleterious effects. Therefore, the developed quantitative multiplex method includes 22 blood neuroinflammatory, brain function-linked, genetic and general state biomarkers for disease state evaluation and monitoring, patient stratification and differential diagnosis purposes.</p><p><strong>Conclusions and Perspectives:</strong> Further analytical validation would be performed to fully assess method performances (stabilities, intra-laboratory reproducibility). Then, large patient cohorts would be analyzed to evaluate and clinically validate this innovative tool and its capacity to differentiate diseases, severity and co-pathologies. Such tool would bring dementia diagnosis within the landscape of personalized medicine fostering better patient care.</p>

News from LabRulezLCMS Library - Week 35, 2024

<p><strong>In the week of September 2, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.</strong></p><blockquote><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></h4></blockquote><blockquote><p><strong>👉 Search and filter in the largest global database</strong> with over <strong>3 500 GC, LC, MS and spectroscopy webinars</strong>.</p></blockquote><h5><strong>1. SCIEX: SCIEX OS software summer school - Data processing: screening</strong></h5><ul><li>Mo, 2.9.2024</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4158">Registration</a></li></ul><p>Join us for our summer school series dedicated to enhancing your proficiency with SCIEX OS software.</p><h5><strong>2. Thermo Fisher Scientific: LC Columns Tips &amp; Tricks</strong></h5><ul><li>We, 4.9.2024 10:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4233">Registration</a></li></ul><p>Our upcoming webinar, LC Columns Tips &amp; Tricks, is designed to help you get the most out of your LC columns.</p><h5><strong>3. Waters Corporation: Learning Advanced Analytical Technologies to Expedite the development of Antibody Drug Conjugates</strong></h5><ul><li>We, 4.9.2024 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4200">Registration</a></li></ul><p>Learn how Light Scattering can benefit your drug development.</p><h5><strong>4. Bruker: &nbsp;LabCast: Spatial profiling to transform molecular pathway awareness using neofleX</strong></h5><ul><li>We, 4.9.2024 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4201">Registration</a></li></ul><p>The multiomics capabilities of MALDI Imaging.</p><h5><strong>5. Technology Networks/SCIEX: Enhancing MS-Based Omics Analysis in a Model Organism</strong></h5><ul><li>We, 4.9.2024 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4199">Registration</a></li></ul><p>How LC-Zeno SWATH DIA outperforms conventional DDA in annotating metabolites and lipids in the model organism Gammarus fossarum.</p><h5><strong>6. SelectScience/Tecan: Streamline your sample preparation with Resolvex® prep</strong></h5><ul><li>We, 4.9.2024 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4181">Registration</a></li></ul><p>Specialists from Eprep will explain how the Resolvex® Prep simplifies chromatography and mass spectrometry sample preparation.</p><h5><strong>7. Thermo Fisher Scientific: Advances in proteoform discovery and identification</strong></h5><ul><li>Th, 5.9.2024 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4218">Registration</a></li></ul><p>Native nano-DESI mass spectrometry imaging of proteins up to 230 kDa: Advances in proteoform discovery and identification.</p>

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News from LabRulezLCMS Library - Week 35, 2024

High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns

Webinars LabRulezLCMS Week 36/2024

News from LabRulezLCMS Library - Week 34, 2024

Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants

Webinars LabRulezLCMS Week 35/2024

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Cumulative Neutral Loss Model for Fragment Deconvolution in Electrospray Ionization High-Resolution Mass Spectrometry Data

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