Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 22nd September 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!<blockquote>👉 <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></blockquote><blockquote>👉 Need info about different analytical techniques? Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library">LabRulezGCMS</a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library">LabRulezICPMS</a> libraries.</blockquote>This week we bring you application notes by Agilent Technologies, Shimadzu and Waters Corporation and poster by Thermo Fisher Scientific / HPLC Symposium!<h3>1. Agilent Technologies: Quantitation of N-Nitroso Nebivolol in Nebivolol Tablet Formulation</h3>Using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+triple+quadrupole">Agilent 6495D triple quadrupole LC/MS system</a><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_nebivolol_tablet_formulation_6495_qqq_lc_ms_system_5994_8053en_agilent_e7f21eb12f.pdf">Full PDF for download</a></li></ul>N-Nitroso Nebivolol is a NDSRI of Nebivolol, a beta-1 adrenergic receptor antagonist commonly used in the treatment of hypertension and heart failure. In the carcinogenic potency categorization published by the FDA, N-Nitroso Nebivolol has been categorized as Class 4, indicating the maximum allowable intake limit for the impurity to be 1,500 ng/day. Nebivolol has maximum daily dosage limit of 40&nbsp;mg for adults.Considering a maximum daily dose of 40&nbsp;mg and allowable intake of 1,500&nbsp;ng/day for the nitrosamine impurity in the formulation under study, the specification limit of N-Nitroso Nebivolol has been calculated as 22.5 ng/mL for the 0.6 mg/mL test concentration used during this study.&nbsp;This application note demonstrates a highly specific, sensitive, and reproducible method for the quantitation of N-Nitroso Nebivolol in a 20 mg Nebivolol tablet formulation. The method uses a 1290 Infinity II LC coupled to a 6495D triple quadrupole LC/MS/MS. The multiple reaction monitoring (MRM) conditions and ion source parameters were automatically optimized using the optimizer tool available with MassHunter Acquisition software to maximize the sensitivity of the target analyte. The API peaks were diverted to waste with the help of an integrated, software-controlled diverter valve during quantitation of the&nbsp;impurity.<h4>Experimental</h4><h5>Equipment&nbsp;</h5>The analysis was performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC">1290 Infinity II LC system</a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+triple+quadrupole">6495D triple quadrupole LC/MS/ MS</a>. The LC system was equipped with the following&nbsp;modules: – Agilent 1290 Infinity II high-speed pump (G7120A) – Agilent 1290 Infinity II multisampler (G7167B) – Agilent 1290 Infinity II multicolumn thermostat (G7116B) – Agilent 1290 Infinity II diode array detector (G7117A)<h4>Conclusion&nbsp;</h4>A highly sensitive and robust MRM method was developed for the quantitation of N-Nitroso Nebivolol in 20&nbsp;mg Nebivolol tablet formulation using an Agilent 6495D triple quadrupole LC/MS system. The chromatographic method developed provided good separation between the analyte and the API. The method showed LOD and LOQ of 2 and 5 pg/mL, respectively. The minimum signal-to-noise ratio at the LOQ level was found to be more than 50:1 (Peak to Peak). The calibration curve from 5 pg/mL to 50&nbsp;ng/mL was found to be linear, with 1/x weighting. R and R2 values were 0.999 and 0.997, respectively. Spike recovery analysis showed the efficiency of sample extraction with a recovery percentage of 116.8% in API, 108.3% in placebo, and 111.8% in tablet formulation at the spiking level of 200 pg/mL. The method was found to be highly reproducible at the LOQ level, while the %RSD value for the area response of seven replicate injections was approximately 3.1%.<h3>2. Shimadzu: Efficient Method Development for Synthetic Peptide and Related Impurities</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_00780_en_4d0aa014ea.pdf">Full PDF for download</a></li></ul><h5>User Benefits</h5><ul><li>LabSolutions MD improves the efficiency of the entire workflow for method development of synthetic peptide and impurities.&nbsp;</li><li>Screening for multiple mobile phases and columns can be automated using mobile phase and column switching valves.&nbsp;</li><li>Molecular weights of peptides and related impurities can be estimated and accurately tracked with LCMS-2050, a single quadrupole mass spectrometer.</li></ul>Peptide therapeutics, characterized by specific amino acid sequences crucial to their function, can be synthesized chemically, similar to small molecule drugs. The production of synthetic peptides involves multiple steps, including deprotection, activation, coupling, and the cleavage of the final sequence from the solid support. Impurities, such as those resulting from premature chain termination or missing amino acids, can affect the safety and efficacy of the final product. Therefore, these impurities must be separated by liquid chromatography (LC). In LC analysis, selecting the appropriate mobile phase and column across a wide range of combinations is critical for achieving optimal separation, as it significantly impacts the separation. However, since the separation pattern varies depending on the peptide chain lengths, amino acid compositions, and presence of modifications, optimizing separation for each peptide sequence is time-intensive. This study describes how to efficiently find the best separation conditions for peptides and related impurities utilizing LabSolutions MD, a dedicated software for supporting method development, through both screening and optimization phases.<h4>Experimental</h4><ul><li>LC System: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+X3">Nexera X3</a> (Method Scouting System)</li><li>MS System: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050">LCMS-2050</a></li></ul><h4>Automated Peak Tracking by LCMS-2050&nbsp;</h4>LC chromatograms obtained with a gradient time of 5 min, flow rate of 0.6 mL/min, and initial concentration of 5 % and 15 %, along with molecular weights for FLP and impurities, are shown in Fig. 18. The UV spectra for each impurity are displayed in Fig. 19. The similarity between UV spectra of Met(O2), p.A1del, p.A1_E2del, p.A1_K3del, and p.A1_D5del is greater than 0.99, suggesting that peak tracking based on UV spectra would be challenging. In contrast, LabSolutions MD enables peak tracking based on molecular weights acquired by LCMS-2050, facilitating accurate identification of compounds with similar UV spectra (Fig. 18). The estimated molecular weights for each compound show small mass errors compared to the theoretical values (Table 3), which can be used to confirm the molecular weights of known compounds as well as to approximate the molecular weights of unknown impurities.<h4>Conclusion</h4>The separation patterns of synthetic peptides vary depending on mobile phase composition, column type, and various LC parameters, such as gradient conditions, column oven temperature, and flow rate. Separation behavior can also differ based on peptide structure, including length, amino acid composition, and the presence of modifications. Therefore, it is necessary to optimize the separation for each peptide sequence individually. However, optimizing analytical conditions through numerous analyses and data processing can be time-consuming. LabSolutions MD automates the entire workflow, including generating analysis schedules, preparing mobile phases, and processing the data, thanks to functionalities such as automated peak tracking, ranking of chromatograms, and design space visualization. This article introduced a case where the optimal separation conditions for synthetic peptides were efficiently identified through screening and optimization phases. LabSolutions MD also offers fully automated gradient optimization by AI algorithm to meet user-defined criteria. For more details, please refer to the application news, “Automatic Optimization of Gradient Conditions by AI Algorithm on Synthetic Peptide and Impurities: 01-00814”.<h3>3. Thermo Fisher Scientific / HPLC Symposium: Sensitive cationic lipids impurities analysis with quantitation by charged aerosol detection and simultaneous mass confirmation by MS</h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_sensitive_cationic_lipids_impurities_analysis_quantitation_po_en_3dacf61554.pdf">Full PDF for download</a></li></ul>Cationic lipid is a critical component in lipid nanoparticle formulations for delivering nucleic acids such as mRNA, siRNA, antisense oligonucleotides etc. [2] Cationic lipids are amphiphilic molecules that possess a hydrophilic region, a hydrophobic region, and a linker structure connecting the two. [1] Charged aerosol detector (CAD) and evaporative light scattering detectors (ELSD) are preferred for characterizing lipid nanoparticle components, with CAD being the better choice for impurity quantification due to its higher sensitivity [3]. In a previous study, an inverse gradient method was developed to quantify impurities in cationic lipids raw materials.[1] To demonstrate the inverse gradient method on CAD HP, the same cationic lipids -- R-DOTAP, DLin-KC2- DMA and ALC-0315 -- were measured to this experiment as in Application note 003384 [1]. New diverter valve and software features were showcased, along with method’s sensitivity and repeatability.<h4>Materials and methods</h4>Using the inverse gradient method from Application note 003384 [1], to demonstrate impurity analysis in cationic lipids on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2176">Thermo Scientific Vanquish Charged Aerosol Detector HP (CAD HP)</a> integrated in the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743">Thermo Scientific Vanquish Flex Inverse Gradient UHPLC System</a>, with parallel mass confirmation by the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/413">Thermo Scientific ISQ EM Single Quadrupole Mass Spectrometer</a>.&nbsp;Methods: The workflow uses a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743">Vanquish Flex Inverse Gradient LC system</a> with Vanquish CAD HP and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1970">Thermo Scientific Hypersil GOLD C8 column</a>.<h5>Data Analysis&nbsp;</h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76">Thermo Scientific Chromeleon Chromatography Data System (CDS)</a> 7.2.10 was used for data acquisition and analysis.<h4>Conclusions&nbsp;</h4>The CAD HP integrates perfectly into the Vanquish Inverse Gradient LC system and offers high sensitivity and excellent repeatability for quantitative cationic lipid impurity analysis, while the ISQ EM MS provides straightforward mass confirmation with high repeatability, all under compliance-ready conditions.<h3>4. Waters Corporation: Quantitative Analysis of Four Long-Chain Lysophosphatidylcholines (LPCs) in Dried Blood Spot using Liquid Chromatography Tandem Mass Spectrometry for Clinical Research</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008826_en_dc154872b6.pdf">Full PDF for download</a></li></ul><h5>Benefits</h5><ul><li>Good chromatographic resolution of all forms</li><li>Short extraction time compared to traditional methods for lipid extraction</li><li>Short analysis injection-to-injection 7 minutes</li></ul>Historically, very long chain fatty acids have been quantified using direct transesterification and gas chromatography methods which involve time-consuming and labor-intensive sample preparation and analysis. Consequently, simpler LC-MS/MS methods have been reported for the measurement of long-chain LPCs (LPCs generic chemical structure, Figure 1) in DBS. Interferences have been reported using positive ESI, thus researchers have developed methodologies utilizing negative ESI to circumvent isobaric interferences when there is lack of chromatographic resolution. Here a UHPLC-MS/MS method is described using acetate adducts for the reliable analysis of four long-chain LPCs in DBS for clinical research.&nbsp;A UHPLC-MS/MS method for quantifying a panel of LPCs (C20:0, C22:0, C24:0, and C26:0-LPCs) was developed. The mobile phase composition consisted of a mixture of water, acetonitrile, and isopropanol with 5 mM ammonium acetate as a modifier. The DBS sample extraction used a working solution prepared with the deuterated LPC internal standards in methanol. The method was developed with an ACQUITY UPLC I-Class PLUS System (Fixed Loop) using an ACQUITY Premier CSH C18 Column, which provided baseline resolution of endogenous isobaric interferences for the saturated forms of the targeted LPCs. The detector was a Xevo TQ-S micro Mass Spectrometer operated in negative ESI mode. This methodology resulted in a total analysis time (injection-to-injection) of 7 minutes.<h5>LC Conditions&nbsp;</h5><ul><li>LC system: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/199">ACQUITY UPLC I-Class PLUS FL&nbsp;</a></li><li>System Detection: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/209">Xevo TQ-S micro Mass Spectrometer&nbsp;</a></li><li>Plate: Waters 96-well Sample Collection Plate, 350 µL&nbsp;</li><li>Column(s): <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1952">ACQUITY Premier CSH C18 Column</a>, 2.1 mm x 50 mm, 1.7 µm (p/n: 186009460)</li></ul><h5>MS Conditions&nbsp;</h5><ul><li>MS system: Xevo TQ-S micro Mass Spectrometer</li></ul><h5>Data Management&nbsp;</h5><ul><li>MS software: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635">MassLynx™ v4.2 Software SCN 1045&nbsp;</a></li><li>Informatics: TargetLynx™ XS v4.2 Software SCN1045 and QUAN Review</li></ul><h4>Results and Discussion&nbsp;</h4>During method development, different types of column chemistry, mobile phase composition, and ESI polarity modes were assessed to optimize chromatographic peak shape, analytical sensitivity and manage interferences. In positive mode, ammonium formate favored the formation of m/z 184 and m/z 104 as daughter ions for all the LPCs. The m/z 184 ion lacks specificity because most phospholipids share this moiety, so the ion m/z 104 was selected as quantifier. Having evaluated conditions in positive mode for the analysis of the four saturated LPCs, it was observed that some existing interferences were not only affecting the parent masses of the analytes but their corresponding internal standard parent ion masses too. Hence, negative ionization using ammonium acetate as modifier was assessed for the detection of LPCs. Acetate adducts [M+CH3COO]- were the most abundant parent ion masses, generated under these conditions for each LPC. The acyl-chain product ions generated in negative ionization reduce the detection of interferences coming from other phosphocholine headgroups. The latter detection conditions were used for the validation of the method presented herein.<h4>Conclusion&nbsp;</h4>An LC-MS/MS clinical research method for measuring C20:0, C22:0, C24:0, and C26:0 LPCs with the ACQUITY UPLC I-Class PLUS FL System, and the Xevo TQ-S micro Mass Spectrometer that:<ul><li>Demonstrates good linearity, with no significant carryover, or matrix effects</li><li>Offers good precision with a total reproducibility and repeatability ≤10% CV</li><li>Offers reliability in the identification of LPCs from interfering isobaric compounds Provides good selectivity and analytical sensitivity in negative mode and is relatively fast as approximately 220 samples can be analyzed in 24 hours</li></ul>

News from LabRulezLCMS Library - Week 39, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 13th October 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!<blockquote>👉 <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></blockquote><blockquote>👉 Need info about different analytical techniques? Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library">LabRulezGCMS</a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library">LabRulezICPMS</a> libraries.</blockquote>This week we bring you application notes by Agilent Technologies, Shimadzu and Waters Corporation and poster by Thermo Fisher Scientific / HPLC!<h3>1. Agilent Technologies: Expanding Coverage for the Analysis of PFAS in Paper-Based Food Packaging Materials</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_pfas_food_contact_materials_6545_lcqtof_5994_8516en_agilent_0d3cbb1f4d.pdf">Full PDF for download</a></li></ul>PFAS are manufactured chemicals used in food packaging for their grease- and waterproof properties. However, PFAS are persistent in the environment and may pose health risks.2 This study presents a workflow to screen for PFAS in FCMs using LC/Q-TOF. The FluoroMatch software suite was used to automate PFAS data annotation of knowns and unknowns as well as generate an interactive visualization dashboard, enhancing analysis accuracy and coverage.&nbsp;The FluoroMatch suite is an open-source set of tools designed to streamline the suspect and nontarget screening of PFAS compounds. It automates several processes, including file conversion, chromatographic peak picking, blank feature filtering, PFAS annotation based on precursor and fragment masses, homologous series detection, compound classification (PFAS versus not PFAS), and reporting annotation confidence. The software library contains 15,643&nbsp;PFAS species and associated fragmentation patterns, with the capability to add more.3&nbsp;Various FCM samples, including takeaway containers and microwave popcorn bags, were rigorously extracted and analyzed. Suspect screening was employed for PFAS quantitation, while nontargeted workflows enabled the identification and semi-quantitation of PFAS that were lacking&nbsp;standards.<h4>Experimental</h4><h5>LC/MS analysis&nbsp;</h5>Extracts were analyzed using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC">Agilent 1290 Infinity II LC system</a> coupled with an Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6545+LC%2FQ-TOF">6545 LC/Q-TOF</a>. An Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=PFC-Free+HPLC+Conversion+kit">PFC-Free HPLC Conversion kit</a> (part number 5004‑0006) and an Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+PFC+delay+column">InfinityLab PFC delay column</a> (part number 5062‑8100) were used to minimize background PFAS levels. Liquid chromatographic separation was performed on a 1.8&nbsp;µm, 2.1 × 100 mm, Agilent ZORBAX RRHD StableBond SB‑C18 column (part number 858700-902) with a corresponding guard column (Agilent part number 821725-902). The mobile phases were (A) water with 20&nbsp;mM ammonium acetate and (B) methanol. The flow rate was 0.4&nbsp;mL/min. The elution gradient was: 5% B (0 to 0.5&nbsp;minutes), linear increase to 40% B (0.5 to 3 minutes), linear increase to 80% B (3 to 16 minutes), hold 80% B (16 to 18 minutes), linear increase to 100% B (18 to 22 minutes), hold 100% B (20 to 22&nbsp;minutes), decrease to 5% B (22 to 22.5 minutes), followed by a 4 minutes post‑run re‑equilibration. The injection volume was 10 μL, and the column temperature was maintained at 50 °C. For the LC/Q‑TOF, the drying gas was set to 4 L/min at 230 °C, the nebulizer to 20 psi, and the sheath gas to 12 L/min at 375 °C.<h4>Results and discussion</h4>PFAS identification using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=FluoroMatch">FluoroMatch</a> Using FluoroMatch Visualizer, interactive tools for visualizing PFAS data were generated, including mass defect plots, accurate mass versus retention time plots, MS/MS fragmentation plots, annotation tables, and fragment screening. Individual homologous series were selected based on nominal mass and normalized mass defect, allowing for the observation of patterns and identification of outliers. This interactive cross-filtering simplified the evaluation of PFAS features and enhanced confidence in nontargeted results.&nbsp;FluoroMatch was used to annotate a homologous series (C3 to C14) of perfluoroalkyl carboxylic acids (PFCAs), including PFHxA (perfluorohexanoic acid), PFHpA (perfluoroheptanoic acid), PFOA (perfluorooctanoic acid), PFNA (perfluorononanoic acid), PFDA (perfluorodecanoic acid), and PFDoDA (perfluorododecanoic acid). This workflow demonstrated that the suspect screening approach successfully identified common PFAS with available standards, while FluoroMatch was able to annotate additional PFAS that were lacking standards.&nbsp;Additionally, FluoroMatch annotated C6 and C8 perfluoroalkyl sulfonic acids (PFSAs), which were not identified by suspect screening. This highlights the value of a hybrid workflow that combines traditional suspect screening with nontargeted tools like FluoroMatch, enhancing confidence in the nontargeted results through their complementary nature. Specifically, FluoroMatch allows for homologous series detection to help annotate incomplete or noisy spectra. FluoroMatch Visualizer evaluation helps incorporate additional lines of evidence to annotation like retention time patterns and Kendrick mass defect to aid in annotation.<h4>Conclusion&nbsp;</h4>This study successfully demonstrates the effectiveness of FluoroMatch software in automating the annotation and visualization of PFAS compounds in food packaging materials. The integration of FluoroMatch Visualizer provides a comprehensive approach to identifying both targeted and nontargeted PFAS compounds. The results highlight the prevalence of PFAS in various paper‑based FCMs, with clamshell to-go boxes showing the highest concentrations, particularly PFOA and PFDA, which exceed European Union regulatory limits. As PFAS can migrate into food, especially at higher temperatures, the pervasiveness of PFAS in FCMs raises concerns about potential exposure through hot meals and microwave heating. The widespread detection of PFAS in clamshell to-go boxes and other FCMs indicates a need for stricter regulations to reduce PFAS use in food packaging to protect consumer&nbsp;health.<h3>2. Shimadzu: An Ultra-high Sensitivity Analysis of 29 PFAS Compounds in Drinking Water by Direct Injection</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_01026_en_dcf554dd6f.pdf">Full PDF for download</a></li></ul>PFAS (Per- and Polyfluoroalkyl Substances) are synthetic fluorinated organic compounds. More than a few thousand compounds exist, each with a varied carbon chain length, functional groups, and structural isomers. Because of its useful qualities, such as their ability to repel water and their nonstickiness, PFAS are found in a wide range of consumer and industrial products, including firefighting foams and coatings. Their high stability and persistence raise concerns about their impact on human health and the environment. In recent years, regulations and studies regarding PFAS have been increasing globally. For example, the U.S. Environmental Protection Agency (EPA) has set a Maximum Contaminant Level (MCL) of 4 ng/L for PFOA and PFOS in drinking water. To improve the analysis of PFAS, more sensitive, accurate and faster methods are needed. This application introduces the ultra-high sensitivity analysis of 29 PFAS in drinking water by direct injection using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8065XE">LCMS-8065XE</a> (Fig. 1) and addresses the demands of testing laboratories for PFAS quantification.<h4>Analytical conditions</h4>The HPLC and MS analytical conditions are shown in Table 1 and 2. The test compounds used were the 29 compounds included in methods EPA 533 and 537.1 for drinking water analysis. The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8065XE">LCMS8065XE</a> is equipped with a new ESI probe (StreamFocus) and a new collision cell (UFsweeper IV), providing more sensitive and accurate results in PFAS analysis. A delay column was installed between the mixer and autosampler to separate PFAS from the HPLC system and those in the sample. To ease the analysis, same type of column is used as delay and analytical columns.<h4>Conclusion&nbsp;</h4><ul><li>29 of PFAS in drinking water were measured with high sensitivity (&lt;1 ng/L) by direct injection method using the LCMS-8065XE.&nbsp;</li><li>Good recovery and reproducibility were obtained in drinking water samples, confirming that this analytical method can deliver accurate quantification for PFASsin drinking water.</li></ul><h3>3. Thermo Fisher Scientific / HPLC: Efficient tandem capillary flow LC-MS with short μPAC columns and a single ionization source</h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_efficient_tandem_capillary_flow_lc_ms_short_pac_columns_po_en_f38e078c9a.pdf">Full PDF for download</a></li></ul>Nano-flow tandem direct injection (TDI) workflows increase sample throughput and instrument productivity with flow rates below 1 µL/min but need dual-column ESI interfacing to maintain chromatographic performance. At flow rates above 1 µL/min with capillary flow columns, post-column dispersion impacts performance less, allowing low-dispersion switching valves in the fluidic path. We demonstrate the Vanquish Neo UHPLC system TDI workflow for capillary flow LC with a high throughput short pillar array column. This dual-column, single-ESI emitter setup reduces variability, extends emitter life through efficient salt management, and ensures high reproducibility with dedicated gradient pumps and intelligent method design.<h4>Materials and methods</h4><h5>Sample Preparation&nbsp;</h5>HeLa cell digests were resuspended in 0.1% TFA and 1% ACN to obtain a 200 ng/µL stock solution, sonicated, diluted, and vortexed before use.<h5>LC-MS configuration&nbsp;</h5>Samples were analyzed using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398">Vanquish Neo UHPLC</a> instrument that was configured in tandem direct injection mode with an additional pump module and a 10 µL injection loop for maximum throughput. Two µPACM Neo High-Throughput Plus columns were positioned between low-dispersion 6-port switching valves in the column compartment. A 20 µm ID nanoViper line connected the alternating eluents to an ESI emitter with an integrated liquid junction, positioned in a Thermo Scientific EASY-Spray source coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/402">Orbitrap Exploris 240 mass spectrometer</a><h4>Conclusions&nbsp;</h4><ul><li>Using optimized µPAC Neo HT Plus columns in combination with the Tandem Vanquish Neo UHPLC system heightened the productivity of bottom-up proteomics measurements to 86 - 92% (gradient-dependent)&nbsp;</li><li>At these throughputs, choice of emitter only has a minor effect on identifications, FWHM and TIC. Decreasing the flow rate to 1 uL/min causes peak broadening and increases ionization efficiency which is reflected in higher TICs. In this study, reducing the flow rate only showed a positive effect on protein IDs for the longest gradient (100 SPD).&nbsp;</li><li>With the optimized methods, identifications up to 4713 protein groups for the 180 SPD and 5692 protein groups for the 100 SPD were achieved. The CVs using the same column were between 5-6% and between two columns at around 7-8% (n=6)&nbsp;</li><li>Column reproducibility was showcased for 12 columns across four different production batches. Overall, the ID rate variation between the columns was 1.4%. The median coefficient of variation of the retention times of spiked-in PRTC peptides across the tested 12 columns was 1.8%</li></ul><h3>4. Waters Corporation: Oligo Mapping of sgRNA Digests: Leveraging Xevo MRT Mass Spectrometer Performance and Streamlining Data Analysis</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009009_en_f35f22bebc.pdf">Full PDF for download</a></li></ul><h5>Benefits&nbsp;</h5><ul><li>Confident characterization of sgRNA digestion products with sub-ppm mass accuracy of oligonucleotide precursors, outstanding MS resolution (~100,000), and sensitivity at high acquisition speed</li><li>A compliance-ready informatics workflow featuring the waters_connect MAP Sequence App (ver 1.0) streamlines oligonucleotide mapping of sgRNA digests</li><li>RapiZyme MC1, a new ribonuclease - offers unique cleavage specificity and the opportunity to generate overlapping digestion products, enabling complete (100%) sequence coverage for sgRNAs</li></ul>Single guide RNAs (sgRNAs) were first described in 20121 when two RNA molecules essential for CRISPR-Cas9- mediated DNA cleavage – the trans-activating CRISPR RNA (tracrRNA), which serves as a scaffold for the Cas9 nuclease, and the CRISPR RNA (crRNA), responsible for DNA target recognition - were fused into a single RNA construct. This fusion created the sgRNA, a critical component of the CRISPR-Cas9 gene editing system.1–2 The sgRNA molecule directs the Cas9 nuclease to introduce precise double-stranded breaks in DNA, enabling targeted genetic modifications.&nbsp;Since its discovery, which was honored with the Nobel Prize in Chemistry, CRISPR technology has transformed gene editing applications. Beyond basic research, it has been adapted for rapid diagnostic tools, such as COVID19 tests developed during the 2020 global pandemic3 , and holds promise for therapeutic interventions in genetic diseases, cancer, and infectious diseases. As sgRNAs are typically synthesized via solid-phase oligonucleotide synthesis, their analytical characterization requires confirmation of both the intact molecular weight4 and the sequence verification to ensure functional accuracy.5&nbsp;Traditional LC-MS workflows for sgRNA digest oligo mapping have been labor-intensive and time-consuming, relying heavily on manual data analysis and interpretation. Recently6 , a UPLC-MS and informatics workflow designed for automated sgRNA sequence mapping was presented following enzymatic digestion with a panel of ribonucleases, including RNase T1, hRNAse 4 and two RNase T2 enzymes: RapiZyme MC1 and Cusativin. This application note extends the effectiveness of this workflow (Figure 1) when implemented on a Xevo MRT Mass Spectrometer (Figure 1), while focusing specifically on MC1 digestion of the Waters™ sgRNA LC-MS standard, where complete unique digested product coverage was obtained.<h4>Experimental</h4><h5>LC Conditions</h5><ul><li>LC-MS system: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148">Xevo MRT</a> (multi-reflecting time-of-flight) Mass Spectrometer coupled with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191">ACQUITY™ Premier UPLC (Binary) System&nbsp;</a></li><li>Column: ACQUITY Premier Oligonucleotide BEH™ C18 Column 300 Å, 1.7 µm, 2.1 x 150 mm, (p/n:186010541)</li></ul><h4>Results and Discussion&nbsp;</h4>An informatics workflow (Figure 1) featuring the waters_connect MAP Sequence App (version 1.0) was used to facilitate automated data processing of UPLC-MSE datasets acquired following the enzymatic digestion of an sgRNA standard with RapiZyme MC1.6–7 The informatics processing workflow consisted of three steps:&nbsp;<ol><li>In-silico digestion: The mRNA Cleaver MicroApp was used to generate predicted, in-silico digested oligonucleotide digestion products, based on the target RNA sequence.&nbsp;</li><li>Data processing and mass matching: The waters_connect MAP Sequence App processed the UPLC-MSE data, matching the predicted neutral monoisotopic masses of digested oligonucleotides to the experimentally acquired MS1 data.&nbsp;</li><li>Sequence coverage visualization: The resulting sequence coverage for the RNA digest was summarized and visualized using the Coverage Viewer MicroApp.</li></ol><h4>Conclusion&nbsp;</h4><ul><li>An informatics workflow, incorporating the waters_connect MAP Sequence App (ver 1.0) was successfully demonstrated for UPLC-MSE digested oligo mapping analysis of sgRNA digests using the Xevo MRT QTOF Mass Spectrometer.&nbsp;</li><li>Confident peak assessments for analysis of sgRNA digests were achieved with the Xevo MRT QTOF Mass Spectrometer, which delivered excellent sensitivity, high resolution MS (100,000), and mass accuracy of less than 1 ppm.</li><li>Complete sgRNA sequence coverage (100%) was achieved by employing RapiZyme MC1, a novel endonuclease introduced by Waters, which provided unique cleavage specificity and predictable missed cleavages to generate longer, unique, overlapping digestion products.</li><li>Oligonucleotide isomeric sequences resulting from enzymatic digestion were effectively differentiated using the waters_connect CONFIRM Sequence App, which matched elevated energy fragment ions patterns to their respective sequences, allowing unambiguous isomer identification.</li></ul>

News from LabRulezLCMS Library - Week 42, 2025

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 20th October 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!<blockquote>👉 <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></blockquote><blockquote>👉 Need info about different analytical techniques? Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library">LabRulezGCMS</a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library">LabRulezICPMS</a> libraries.</blockquote>This week we bring you posters by Agilent Technologies / AOAC, Shimadzu / AOAC and Thermo Fisher Scientific / HPLC Symposium and application note by Waters Corporation!<h3>1. Agilent Technologies / AOAC: Evaluating System Robustness of a High-Sensitivity Triple Quadrupole LC/MS for PFAS Analysis in Food Matrix Over an Extended Period&nbsp;</h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ps_pfas_salmon_6495d_robustness_aoac_2025_t004_en_agilent_9b7b469a22.pdf">Full PDF for download</a></li></ul>Due to the rapidly evolving regulatory environment of per- and polyfluoroalkyl substances (PFAS), many analytical laboratories are opting to use highsensitivity triple quadrupole mass spectrometers for the targeted analysis of such compounds to ensure meeting present and potential future requirements for sensitivity and PFAS coverage. These assays are regularly conducted in a high-throughput manner involving complex matrices such as food, soil, wastewater or biological samples. Therefore, system robustness and instrument uptime are crucial for such applications to maintain productivity and profitability. In this work, we present results from an expedited robustness testing of a high-sensitivity triple quadrupole LC/MS system using select PFASs in one of the most challenging food matrices.<h4>Experimental</h4><h5>Instrumentation</h5>Data was collected using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Agilent+1290+Infinity+II+LC">Agilent 1290 Infinity II LC</a> system coupled to an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+LC%2FTQ">Agilent 6495D LC/TQ mass spectrometer.</a> Instrument parameters are summarized in Tables 1 and 2.<h4>Conclusions</h4>The robustness of a 6495D LC/TQ was tested using undiluted salmon extract, a complex and challenging food matrix. During the experiment, the system was exposed to more than 130 mL of the matrix over a period of ~3 weeks of continuous operation, without any maintenance. System performance was monitored by injections of a mixture of native and isotopically labelled PFAS samples. The monitored signals exhibited low variations (RSD&lt;6%) throughout the entire experiment. No obvious performance degradation or significant contamination of the inner ion optics elements were observed after the conclusion of the test.&nbsp;Although no loss of instrument performance was observed during this expedited robustness test, a routine maintenance of ion source and optics is recommended to ensure prolonged instrument operation.<h3>2. Shimadzu / AOAC: Analysis of Chlordane by DUIS (Dual Electrospray/Atmospheric Pressure Chemical Ionization) Liquid Chromatography Mass Spectrometry&nbsp;</h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/aoac_2025_w050_036db56f51.pdf">Full PDF for download</a></li></ul>The legalization of cannabis has led to many laboratories developing methods to meet local cannabis testing regulatory requirements. One of the challenging residual pesticides to quantitate is chlordane, an organochlorine compound used as a pesticide on crops until 1983 and for termite treatment in homes until 1988. Chlordane is highly persistent in the environment and has been linked to cancers. Gas chromatography mass spectrometry and atmospheric pressure chemical ionization (APCI) – liquid chromatography mass spectrometry (LCMS) methods were published for analysis of chlordane. However, for laboratories without APCI functionality or who prefer not to switch between two different ionization sources, chlordane remains a challenging target for LCMS analysis. This study demonstrates the successful quantitation of chlordane using additive-free DUIS electrospray ionization – LCMS at concentrations below the New York State testing limits.<h4>Methods&nbsp;</h4>Technical chlordane (CAS 57-74-9) was obtained from Restek, diluted in water, and analyzed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8060">Shimadzu LCMS-8060</a> triple quadrupole mass spectrometer equipped with a DUIS ionization source. The DUIS source was utilized in APCI mode. Technical chlordane is a mixture of transchlordane, cis-chlordane, and related chemicals. The LC parameters shown in Table 1 allowed separation of the cis- and trans-chlordane isomers.<h4>Results&nbsp;</h4>Initial MS data was collected in Q1 and Q3 Scan mode. Due to the 8 chlorine atoms per chlordane molecule, a distribution of isomers is expected in the mass spectrum. LabSolutions Insight Explore was used to predict isotopic distributions for chlordane with and without adducts. The predicted spectra for the [M-H]- and [M+CH3OH]- are shown in Figure 1A and 1B, respectively. The experimentally observed isotopic distributions for the [M-H]- and [M+CH3OH]- ions are shown in Figure 1C and 1D, respectively. Figure 1E shows the relative intensity of the [M+CH3OH]- versus the [M-H]- isotope clusters. As the [M+CH3OH]- showed the greatest intensity, it was selected for optimization.Voltage optimization was performed for the 441.80 to 441.80 MRM with the CID gas turned off. Even after voltage optimization, the signal-to-noise from the primary species in the technical chlordane (in this case, trans-chlordane) was insufficient. Automated source optimization using LabSolutions MRM Connect was used to improve signal by optimizing Interface Temperature, Heating Gas Flow, Nebulizing Gas Flow, Drying Gas Flow, DL Temperature, Heat Block Temperature, and Interface Voltage. As a representative example, Figure 3 shows the impact of Interface Temperature on the MRM intensity for the 441.80 m/z.<h3>3. Thermo Fisher Scientific / HPLC Symposium: HPLC transfer and optimization of deoxycholic acid analysis using HPLC – Charged Aerosol Detector</h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_hplc_transfer_optimization_deoxycholic_acid_analysis_po_en_55bc47dc90.pdf">Full PDF for download</a></li></ul>The United States Pharmacopeial Convention monograph outlines a high-performance liquid chromatography with charged aerosol detection (HPLC-CAD) method [1] for the accurate measurement of deoxycholic acid (also known as desoxycholic acid) and its primary impurity, cholic acid. Key detector settings that influence detection performance metrics such as detection limit, resolution, and linearity include:&nbsp;<ul><li>The evaporation temperature (EvapT) for setting the temperature of the evaporation tube and influencing sensitivity related to volatility factors.&nbsp;</li><li>The filter constant for balancing noise and adjusting the detector's response time to changes in analyte concentration.&nbsp;</li><li>The PV for optimizing the linear range within the detector's dynamic range for a given application.&nbsp;</li></ul>These parameters are crucial for successful method transfer and optimization. Consequently, this work offers guidelines for method transfer and optimization from the Vanquish CAD to the Vanquish CAD HPMethods: A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743">Thermo Scientific Vanquish Flex UHPLC system</a> equipped with both the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2176">Vanquish Charged Aerosol Detector </a>and HP was used to perform method transfer and optimization for deoxycholic acid analysis. Chromatographic separation was achieved using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2005">Thermo Scientific Acclaim 120 C18 column</a> under gradient elution with formic acid-modified aqueous and organic phases<h5>Chromatography Data System&nbsp;</h5>The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76">Thermo Scientific Chromeleon 7.3.2 Chromatography Data System (CDS) software</a> was used for data acquisition and analysis.<h4>Conclusions&nbsp;</h4>This study highlights the successful optimization and method transfer of the USP-NF deoxycholic acid assay using Vanquish CAD detectors. Through careful evaluation of chromatographic performance, detector settings, and calibration parameters, the Vanquish CAD HP was shown to deliver enhanced performance across multiple criteria:&nbsp;<h5>Key Takeaways:&nbsp;</h5><ul><li>Excellent Sensitivity and Response: Vanquish CAD HP demonstrated significantly lower LOD/LOQ values and higher peak responses compared to Vanquish CAD, making it the detector of choice for low-level analyte quantification.&nbsp;</li><li>Enhanced method optimization Flexibility: Vanquish CAD HP allows simultaneous data collection for multiple PVs, ensuring faster method development and optimization.&nbsp;</li><li>Seamless Method Transfer: Transitioning from Vanquish CAD to CAD HP was achieved by optimizing power value (PV), EvapT, and signal filter settings, ensuring improved performance.&nbsp;</li></ul>Together, these findings demonstrate that the Vanquish CAD HP not only supports efficient method transfer but also elevates performance, sensitivity, and usability—making it an ideal solution for modern HPLC applications.<h3>4. Waters Corporation: Multi-Residue Analysis of Pharmaceuticals, Personal Care Products (PPCPs) and Pesticides in Water by Direct Injection Using LC-MS/MS</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008819_en_4c0e9bc91e.pdf">Full PDF for download</a></li></ul><h5>Benefits&nbsp;</h5><ul><li>Sensitivity of Xevo TQ Absolute allows for the trace level detection of PPCPs and pesticides in drinking and surface waters without the need for lengthy clean-up or concentration steps</li><li>The direct injection approach increases productivity and provides faster turn-around time for reporting of results while being well aligned with green analytical chemistry principles</li><li>More than 150 analytes reach limits of quantification of 10 ng/L, making this method suitable for screening for multi-class analytes in water matrices&nbsp;</li></ul>In recent years, there has been increasing concern about the presence of pesticides, pharmaceuticals and personal care products (PPCPs) in water bodies throughout the world.1 The effect of these emerging contaminants on human health and their potential impact on the environment is not yet fully understood. The updated Drinking Water Directive entered into force in January 2021 and is the EU's main law for drinking water. The Directive applies to all water, either in its original state or after treatment, intended for drinking, cooking, food preparation or other domestic purposes, regardless of its origin and whether it is supplied from a distribution network, supplied from a tanker or put into bottles or containers.2 It further protects human health thanks to updated water quality standards, in accordance with World Health Organisation (WHO) recommendations, addressing emerging pollutants, such as endocrine disruptors, and promoting a preventive, risk-based approach to reduce pollution at its source.Water bodies such as rivers, lakes and groundwaters have their status defined in the EU Water Framework Directive.3 An amendment to Directive 2013/39/EU sets environmental quality standards for hazardous substances that require monitoring in these waters. A Commission Implementing Decision4 document, 2022/1307, includes Watch List analytes with Maximum Acceptable Method Detection Limits for compounds of concern across multiple chemical classes. Numerous studies have demonstrated that PPCPs are present at parts-per-trillion (PPT) levels in rivers and streams highlighting the need for methods that are able to detect compounds at these trace levels.5-10 In addition to the low-level detection of these compounds, a major analytical challenge lies in the wide chemical diversity of chemicals of concern. Furthermore, to establish a more complete understanding of water pollution it is necessary to monitor a wide variety of types of water. This application note demonstrates the direct injection, separation and detection of PPCPs and pesticides including acidic, basic, and neutral compounds from multiple regulated analyte lists in a variety of water types.<h4>Experimental</h4><ul><li>LC system: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191">ACQUITY Premier</a> FTN</li><li>MS system: <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/554">Xevo TQ Absolute</a></li></ul><h4>Conclusion&nbsp;</h4>The updated EU Drinking Water and Water Framework Directives provide the regulatory framework for effective monitoring and management of water quality and include provisions for emerging contaminants. Ensuring compliance with these regulations and comprehensive coverage for an ever increasing analyte lists requires versatile, robust analytical methods and high performance LC/MS/MS systems. The direct-injection method investigated in this work is effective at detection of a wide range of PPCPs and pesticides at trace levels suitable for monitoring multiple water types for the presence of compound classes known or suspected to pose risks to human and environmental health. It further provides more rapid turn around time and greener operation than methods that rely upon high volumes of solvent and extensive sample preparation. Method performance was assessed using criteria described in ISO 21253–2:2019. The calibration characteristics, linearity, and residuals were excellent over the concentration range studied. Accuracy and precision were evaluated at three matrix QC levels with six replicate spikes and found to provide excellent performance for 154 analytes in tap and bottled water and 74 analytes in surface water matrix.

News from LabRulezLCMS Library - Week 43, 2025

Ergot alkaloids (EAs) are common contaminants in cereals such as rye, wheat, and barley, posing food safety challenges worldwide. Their quantification, according to EN 17425:2021, relies on HPLC-MS/MS analysis of six primary EAs and their epimers. However, accurate quantification is often hindered by complex food matrices and the lack of suitable internal standards.To overcome these limitations, a semisynthetic approach was developed to produce stable isotope-labeled analogs of all major EAs. Building on previous work, native ergotamine was N6-demethylated and remethylated using ^13CD₃-iodomethane. The resulting labeled standards were validated against native compounds using HPLC-HR-MS/MS, exhibiting identical chromatographic and mass spectral properties. These new standards significantly improve accuracy, reproducibility, and comparability of EA analyses, strengthening food safety monitoring.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.5c03345">Semisynthesis of Stable Isotope-Labeled Ergot Alkaloids for HPLC-MS/MS Analysis</a></h4>Sven-Oliver Herter, Hajo Haase, and Matthias Koch*J. Agric. Food Chem. 2025, 73, 29, 18412–18419<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.jafc.5c03345">https://doi.org/10.1021/acs.jafc.5c03345</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Ergot alkaloids (EAs) are a group of naturally occurring toxic alkaloids produced by fungi of the genus Claviceps, with Claviceps purpurea being the most prevalent in Europe. <a href="javascript:void(0);">(1)</a> They grow on various cereals, but primarily on rye, wheat, and triticale. <a href="javascript:void(0);">(2,3)</a> When a floret is infected with a spore of ergot fungi, the spore mimics a pollen grain growing into the ovary during fertilization, thus destroying the plant ovary and connecting to the vascular bundle of the plant. The ergot fungi produce asexual spores that are released with a sugary honeydew and dispersed by insects to other florets. In preparation for winter, a hardened mycelium (sclerotia) is formed inside the husk of the floret, and alkaloids and lipids are accumulated in the sclerotium. <a href="javascript:void(0);">(4)</a> The harvest of infected cereal leads to the introduction of sclerotia into the food chain. Mechanical techniques for the removal of sclerotia from grain are effective; however, they do not eliminate contamination completely. Residual sclerotia, in the form of dust or fragments, are ground with grain to flour and thereby introduce toxic EAs into foodstuffs. <a href="javascript:void(0);">(4,5)</a> A defining characteristic of EAs is the tetracyclic ring structure, known as ergoline (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.5c03345#fig1">Figure 1</a>). Naturally occurring EAs diverge structurally from ergoline due to the methylation of the N6-atom and various substituents attached to the C8-atom. <a href="javascript:void(0);">(6)</a> Additionally, most EAs possess a double bond between the C9- and C10-atoms in the ergoline structure. <a href="javascript:void(0);">(7)</a> They can be divided into three different groups based on their structural characteristics: lysergic acid and its simple amides, the ergopeptines, and the ergoclavines. <a href="javascript:void(0);">(6)</a> They all share an R-configuration at the C8-position of the ergoline structure. Epimerization at the C8-atom of simple lysergic acid amides and ergopeptines via keto–enol tautomerism is observed when these EAs are exposed to heat, UV light, or high/low pH. <a href="javascript:void(0);">(8,9)</a> However, ergoclavines, such as lysergine and lysergole, do not undergo epimerization due to the absence of a carboxyl group at the C8-atom.In parallel with the establishment of the maximum levels, the European Standard Method (ESM) EN 17425 for the determination of ergot alkaloids in cereals and cereal-based products was published. <a href="javascript:void(0);">(14)</a> The method describes the extraction of EAs from cereals and the subsequent cleanup of the extract using dispersive solid-phase extraction. The determination of EAs is performed with high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). However, the European Committee for Standardization (CEN) has indicated that the method cannot be considered satisfactory for levels below 24 μg/kg for the sum of the priority EAs due to an increase in interlaboratory uncertainties observed during the method validation study. <a href="javascript:void(0);">(14)</a> HPLC-MS/MS is susceptible to various forms of interference when used for quantification. A common challenge is the effect of coeluting molecules on the ionization yield of the analyte molecules. These matrix-dependent signal suppression or enhancements impact the analyte signal and cause the measured concentration to be different from its true value. <a href="javascript:void(0);">(15,16)</a> To overcome this problem, matrix-matched calibration can be employed. This involves the use of a calibration solution whose matrix matches the sample matrix, except for the analyte. However, these specific matrices are rarely available in environmental and food analytical studies. Alternatively, an internal standard (ISTD) can be used to reduce the impact of the matrix on the analyte signal. A suitable ISTD has comparable physio-chemical properties to the analyte, e.g., retention time, ionization response, and fragmentation pattern. Because of the need for an appropriate ISTD for each analyte of interest, this approach is costly and time-consuming. Therefore, in HPLC-MS/MS, ISTDs are typically stable isotope-labeled analogs (2H, 13C, and 15N) of the analyte. <a href="javascript:void(0);">(17)</a> For EAs, only ergometrine-13CD3 and its epimer ergometrinine-13CD3 are currently commercially available, due to their less complex structure compared to the ergopeptides. <a href="javascript:void(0);">(15,18)</a> However, a comprehensive set of all isotopically labeled priority EAs is required to enhance the current ESM and thus to reliably control the EA maximum level in food for infants and young children at 20 μg/kg. In our previous work, we described a straightforward two-step semisynthesis of the ergopeptines 13CD3-ergotamine and 13CD3-ergotaminine, starting from native ergotamine. <a href="javascript:void(0);">(19)</a> The general approach for the two-step semisynthesis of isotopically labeled EAs is illustrated in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.5c03345#fig2">Figure 2</a>. In the first step, native EA is demethylated at the N6-position via an iron-catalyzed dealkylation reaction to yield the corresponding Norergot alkaloid (Nor EA). Subsequent methylation at the N6-atom with an isotopically labeled methylating reagent, e.g., iodomethane, yields the isotopically labeled EA.Given that all priority EAs share the methyl group at the N6-atom, we investigated the possibility of synthesizing all isotopically labeled EAs via a two-step synthesis.<h4>Materials and Methods</h4><h5>HR-MS/MS Measurements</h5>High-resolution tandem mass spectra (HR-MS/MS) were measured on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=55&amp;search=TripleTOF+6600">Quadrupole time-of-flight 6600 mass spectrometer</a> (Sciex, Darmstadt, Germany). The purified solution, comprising either the native EA or Nor EA, was injected directly into the mass spectrometer via a syringe pump for analysis. For HPLC-HR-MS/MS analysis, the QTOF was coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II">1290 Infinity II</a> system (Agilent, Waldbronn, Germany). For the measurements, a Phenomenex Gemini C6–Phenyl (150 × 2.0 mm; 3 μm) column was used. For the MS/MS experiments, an inclusion list with the exact m/z ratio [M + H]+ for the native and isotopically labeled EAs was created. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jafc.5c03345/suppl_file/jf5c03345_si_001.pdf">Table S6</a> presents the QTOF parameters, and <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jafc.5c03345/suppl_file/jf5c03345_si_001.pdf">Table S7</a> presents the HPLC parameters that were used for the HPLC-HR-MS/MS measurement. The predicted sum formulas for the most abundant product ion m/z ratios were calculated using a threshold of ±5 ppm deviation (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jafc.5c03345/suppl_file/jf5c03345_si_001.pdf">Tables S9–S14</a>).<h4>Results</h4><h5>HR-MS/MS of Norergot Alkaloids</h5>The successful N6-demethylation of the EAs to the Nor EAs was confirmed by HR-MS/MS. Purified samples containing either the native EA or the associated Nor EA were introduced into the QTOF mass spectrometer for measurement via direct infusion. The HR-MS/MS spectra of the native EAs and Nor EAs were compared, and specific fragment ions were assigned to the major peaks in the mass spectra. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.5c03345#fig4">Figure 4</a> shows the comparison of the fragment spectra of native ergocristine and the demethylated product norergocristine. The HR-MS/MS spectra of ergocristine coincide with previously reported data in the literature. <a href="javascript:void(0);">(20,21)</a> Upon comparison of the spectra of ergocristine and norergocristine, a similar fragmentation pattern is evident. The observed differences in the intensities of certain fragment ions can be attributed to the lower energy required for the fragmentation of norergocristine. For fragment ions containing the N6-atom, a shift in the m/z ratio for norergocristine compared to ergocristine was observed. This m/z shift corresponds to the loss of the methyl group at the N6-atom and is observed for the fragment ions of ergocristine/norergocristine with the m/z ratios of 348.1711/334.1545, 268.1451/254.1280, and 223.1234/209.1073. The differences in the m/z are 14.0166, 14.0171, and 14.0164, respectively, and match with the m/z ratio of the neutral loss of CH2, which is 14.0157 (cf. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.5c03345#fig4">Figure 4</a>).&nbsp;<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Agric_Food_Chem_2025_73_29_18412_18419_Figure_4_Positive_ESI_HR_MS_MS_spectra_M_H_of_a_ergocristine_and_b_norergocristine_3403f0b9e7.jpg" alt="J. Agric. Food Chem. 2025, 73, 29, 18412–18419: Figure 4. Positive ESI-HR-MS/MS spectra [M + H]+ of (a) ergocristine and (b) norergocristine. In addition to the measured accurate m/z ratio, a suggestive structure is provided for the precursor ion and the major fragment ions along with their calculated exact m/z ratio." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Agric_Food_Chem_2025_73_29_18412_18419_Figure_4_Positive_ESI_HR_MS_MS_spectra_M_H_of_a_ergocristine_and_b_norergocristine_3403f0b9e7.jpg 245w," sizes="100vw" width="2078" height="1106">The fragmentation of the amide bond between the lysergic acid moiety and the cyclic tripeptide corresponds to the fragment ions with m/z ratios of 223.1234/209.1070 and 268.1451/254.1280. Fragmentation of the cyclic tripeptide ring results in the fragment ions with m/z ratios of 348.1711/334.1545. The fragment ion with an m/z ratio of 325.1546 is indicative of the fragment ion of the tricyclic peptide. Given that no modifications were introduced to the cyclic tripeptide ring structure, the spectra of ergocristine and norergocristine both exhibit this specific fragment ion. The HR-MS/MS spectra for the Nor EAs of ergometrine, ergocornine, ergotamine, α-ergocryptine, and ergosine compared to the native EAs are presented in the Supporting Information, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acs.jafc.5c03345/suppl_file/jf5c03345_si_001.pdf">Figures S7, S9, S11, S13, S15</a>. The related structure of all investigated ergopeptines results in a comparable fragmentation pattern, similar to the one observed for ergocristine and norergocristine. In contrast, the representatives of the lysergic acid derivatives, ergometrine and norergometrine, exhibit a less complex product ion spectrum. Based on the measured accurate m/z ratios, we were able to make a structural proposal for all major product ions of the EAs and Nor EAs.<h4>Synthesis of Stable Isotope-Labeled Ergot Alkaloids</h4>To verify the successful synthesis of all 12 isotopically labeled priority EAs, they were spiked at a concentration of 25 ng/mL to a standard with a concentration of 25 ng/mL containing all unlabeled native EAs in acetonitrile and analyzed via HPLC-HR-MS/MS. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.5c03345#fig5">Figure 5</a> depicts the extracted-ion chromatogram (XIC) for the specific fragment ion of the native (m/z 223.1235) and isotopically labeled (m/z 227.1458) EAs. <a href="javascript:void(0);">(22)</a><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Agric_Food_Chem_2025_73_29_18412_18419_Figure_5_Extracted_ion_chromatogram_M_H_XIC_of_a_25_ngm_L_standard_70f2a2760a.jpg" alt="J. Agric. Food Chem. 2025, 73, 29, 18412–18419: Figure 5. Extracted-ion chromatogram [M + H]+ (XIC) of a 25 ng/mL standard for (a) 12 native priority EAs and (b) 12 stable isotope-labeled priority EAs. An inclusion list containing the exact m/z values of each native and stable isotope-labeled EA [M + H]+ was created, and the sample was analyzed in parallel reaction monitoring (PRM) mode on a QTOF mass spectrometer." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Agric_Food_Chem_2025_73_29_18412_18419_Figure_5_Extracted_ion_chromatogram_M_H_XIC_of_a_25_ngm_L_standard_70f2a2760a.jpg 245w," sizes="100vw" width="2079" height="1198"><h4>Discussion</h4>The quantification of EAs in complex food matrices using HPLC-MS/MS is challenging, primarily due to matrix effects and the lack of stable isotope-labeled standards. To address this gap, a semisynthetic approach was developed that targets the N6-atom of the lysergic acid moiety, a structural feature shared among all investigated native priority EAs.The demethylation of the N6-atom to the corresponding Nor EAs was achieved via an iron-catalyzed reaction, and the structure of the Nor EAs was confirmed by HR-MS/MS. A structural assignment for the most intense product ions of the Nor EAs was proposed based on the measured accurate m/z ratios and correlated with specific HR-MS/MS fragments of the native compounds. The characteristic loss in the m/z value associated with the demethylation was observed only for fragment ions of the Nor EAs that contain the N6-atom, thereby confirming successful and selective demethylation for all investigated EAs.For the subsequent remethylation of the Nor EAs, 13CD3-iodomethane was utilized and resulted in the formation of the isotopically labeled C8-R and C8-S epimers of the respective EAs. Overall yields ranged from 8.1% for 13CD3-ergocristine/-inine and up to 29.5% for 13CD3-ergocryptine/-inine. Due to the limited availability of starting material, no yield could be determined for 13CD3-ergometrine/-inine and 13CD3-ergosine/-inine. The epimer ratio (R%:S%) of the ergopeptines ranged between 58:42 for 13CD3-ergocornine/-inine and 45:55 for 13CD3-ergocristine/-inine. Although the synthesis started from a compound with the 8R-configuration, both stable isotope-labeled epimers were obtained in approximately equal amounts. This outcome is favorable as it provides sufficient quantities of both stable isotope-labeled epimers from a single epimeric pure starting material. In contrast, the simpler lysergic acid amide 13CD3-ergometrine/-inine showed less epimerization, with an epimer ratio of 69:31. The structures of the synthesized labeled EAs were confirmed using HPLC-HR-MS/MS. Therefore, a mixture containing all 12 native priority EAs and their corresponding labeled analogs was analyzed. As anticipated for deuterium-labeled compounds, the isotopically labeled EAs showed slightly shorter retention times (1–3 s) compared to their native counterparts. The precursor and fragment ions containing the isotopically labeled methyl group at the N6-position exhibited a shift in the m/z ratio of 4 Da. In contrast, fragment ions without this structural feature were present in the MS/MS spectra of both labeled and unlabeled EAs. With the measured accurate m/z ratios of the precursor and product ions, we were able to predict a sum formula and match specific fragment ions for each major signal in the HR-MS/MS spectrum. Due to the limited availability of the native EAs, only small amounts of the 13CD3-labeled analogs were synthesized, which precluded structural elucidation by NMR. However, the identical fragmentation patterns and signal intensities between labeled and unlabeled EAs, in conjunction with the same retention time in HPLC, provided unambiguous confirmation of the structures for all 12 synthesized stable isotope-labeled priority EAs.For the first time, the procedure described herein enabled the semisynthesis of a complete set of stable isotope-labeled priority EAs. In future work, these standards will be implemented in the ESM and facilitate an enhanced methodology for the quantification of these mycotoxins in food and feed.

Semisynthesis of Stable Isotope-Labeled Ergot Alkaloids for HPLC-MS/MS Analysis

News from LabRulezLCMS Library - Week 48, 2025

Six new pyranonaphthoquinone derivatives, gunacin A–E, together with known gunacin and the isocoumarin (+)-orthosporin, were isolated from the fungus Exobasidium sp. Their structures were elucidated using X-ray crystallography, detailed NMR analysis including ROESY, and mass spectrometry. Two compounds showed remarkable potency against Leishmania mexicana and multiple Trypanosoma species, with EC₅₀ values as low as 0.02–0.24 μM—on par with clinically used antiparasitic drugs.In addition to their antiprotozoal effects, several gunacins demonstrated antibacterial and moderate antifungal activity, and exhibited a broad range of cytotoxicity toward mammalian cell lines. This study is one of the first to explore the chemical potential of Exobasidium, a genus of phytopathogenic Basidiomycota fungi, and highlights smut fungi as an underexplored source of bioactive natural products. The potent activity of gunacins suggests promising opportunities for developing new antiprotozoal therapeutics.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/full/10.1021/acsomega.5c01325">Gunacins: Novel Benzo[g]chromene Derivatives from the Fungus Exobasidium sp. and Their Potent Anti-Leishmania and Trypanosoma Activities</a></h4>Eva Stodůlková, Dominik Lovás, Miroslav Flieger, Alena Zíková, Jakub Zápal, Martin Štícha, Ivana Císařová, Jan Černý, Valéria Grobárová, Martina Slapničková, Tomáš Vomastek, Zuzana Klímová, Marek Kuzma, Jaroslav Semerád, Tomáš Cajthaml, Eva Cséfalvay, Winnie Cherotich Maritim, Adéla Wennrich, Marc Stadler, Tereza Ježková, Andrej Jašica, and Miroslav Kolařík*ACS Omega 2025, 10, 22, 23222–23234<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.5c01325">https://doi.org/10.1021/acsomega.5c01325</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Pyranonaphthoquinones are a diverse and widespread group of secondary metabolites found in plants, <a href="javascript:void(0);">(1,2)</a> fungi, <a href="javascript:void(0);">(3,4)</a> and bacteria <a href="javascript:void(0);">(5,6)</a> with the majority possessing the 3,4-dihydro-1H-benzo[g]chromene-5,10-dione structural motif. Only a few pyranonaphthoquinones feature the 3,4-dihydro-2H-benzo[g]chromene-5,10-dione skeleton, including α-lapachone, constituents of “Lapacho tea”, used in traditional herbal medicine and believed to have anticancer effects. <a href="javascript:void(0);">(7,8)</a> Other examples include rhinacanthin A and its derivatives from plant Rhinacanthus nasutus, <a href="javascript:void(0);">(9)</a> α-caryopterone from Caryopteris clandonensis, <a href="javascript:void(0);">(10)</a> and gunacin (1) isolated from the fungus Ustilago sp. <a href="javascript:void(0);">(3)</a> Lapachones and rhinacanthins exhibit a wide range of biological activities, including cytotoxic, <a href="javascript:void(0);">(9,11)</a> antibacterial, <a href="javascript:void(0);">(1,12)</a> antifungal <a href="javascript:void(0);">(13,14)</a> and antiprotozoal effects. <a href="javascript:void(0);">(1)</a>Exobasidium species <a href="javascript:void(0);">(15)</a> (Exobasidiales, Ustilaginomycotina, Basidiomycota) are worldwide distributed biotrophic plant pathogens, almost infesting plants within the Ericales order. <a href="javascript:void(0);">(16)</a> The genus Exobasidium causes notable economic losses in tea and blueberry production. <a href="javascript:void(0);">(17,18)</a> Despite their prevalence and importance, there have been very few studies on secondary metabolites produced by Exobasidium species. Moreover, a study describing the isolation of scopoletin and scopolin fails to distinguish whether the secondary metabolites originate from the plant host or the fungus itself. <a href="javascript:void(0);">(19)</a> The only credible publications have described the production of auxins, 2-(1H-indol-3-yl) acetic acid, ethyl 2-(1H-indol-3-yl) acetate, <a href="javascript:void(0);">(20)</a> and (S)-2-hydroxy-3-phenylpropanoic acid, <a href="javascript:void(0);">(21)</a> by axenic Exobasidium cultures.This study represents the first comprehensive analysis of Exobasidium secondary metabolites, their isolation, structure determination, and biological activities.<h4>2. Results and Discussion</h4><h5>2.1. Structure Elucidation</h5>In this study, we identified six new pyranonaphthoquinone derivatives, gunacin A–E (2–7), along with the known compounds gunacin (1) and the isocoumarin derivative (+) orthosporin (8) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/full/10.1021/acsomega.5c01325#fig1">Figure 1</a>). Gunacin (1) (2R,3S,4R)-3,6-dihydroxy-8-methoxy-2-methyl-5,10-dioxo-3,4,5,10-tetrahydro-2H-benzo[g]chromen-4-yl acetate was obtained as orange-red needles which crystallized from the MeOH/CH2Cl2 mixture. With the molecular formula C17H16O8, determined by positive high-resolution electrospray ionization mass spectrometry (HRESIMS), data showed a protonated ion [M + H]+ at m/z 349.0920 (calcd for C17H16O8+, 349.0918).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_22_23222_23234_Figure_1_List_of_isolated_compounds_9cc7c55e6c.jpg" alt="ACS Omega 2025, 10, 22, 23222–23234: Figure 1. List of isolated compounds." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_22_23222_23234_Figure_1_List_of_isolated_compounds_9cc7c55e6c.jpg 239w," sizes="100vw" width="1399" height="914"><h5>2.2. Biological Activity</h5>The antimicrobial activity of compounds 1, 3, 5, 6, and 7 was evaluated against model microorganisms, including Escherichia coli, Kocuria rhizophila, Cryptococcus neoformans, and Candida albicans. All tested compounds demonstrated better inhibitory activity against bacteria compared with the positive control, chloramphenicol. The activity against yeasts was approximately on par with the positive control, cycloheximide, except for 3, which showed no inhibitory activity even at the highest tested concentration of 66.7 μM (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/full/10.1021/acsomega.5c01325#tbl5">Table 5</a>).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_22_23222_23234_Table_5_Minimal_Inhibition_Concentration_MIC_of_Compounds_Gunacin_fc54aa5e02.jpg" alt="ACS Omega 2025, 10, 22, 23222–23234: Table 5. Minimal Inhibition Concentration (MIC) of Compounds Gunacin (1), Gunacin A (2), Gunacin B1 (3), Gunacin B2 (4), Gunacin C (5), Gunacin D (6), and Gunacin E (7) against Model Pathogenic Microorganisms E. coli, K. rhizophila, C. neoformans, and C. albicansa" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_22_23222_23234_Table_5_Minimal_Inhibition_Concentration_MIC_of_Compounds_Gunacin_fc54aa5e02.jpg 226w," sizes="100vw" width="1115" height="770">In terms of the morphological effects, HeLa cells and human fibroblasts were examined. The efficacy of 5 on primary fibroblasts is evidenced by the complete disappearance of the MitoTracker Red CMXRos mitochondrial signal at a concentration of 5 μM, which is indicative of mitochondrial dysfunction, particularly with regard to the mitochondrial membrane potential. At higher concentrations, the cells lose their adherence to the surface. At lower concentrations, the actin cytoskeleton remains unaffected, showing the compound’s selectivity for mitochondria. In contrast, HeLa cells are more resistant, with effects on mitochondrial physiology only observed at the highest tested concentration of 125 μM, while the actin skeleton remains unchanged. Compound 1 has similar activity as 5, HeLa cells are significantly more resistant, with notable alterations in cellular morphology only at 125 μM. Effects on fibroblasts were observed at 5 μM, including a loss of polymerized actin and mitochondrial signal. Compound 7 did not show significant toxicity even at 125 μM on Hela cells, except for a slight decrease in the green signal for actin. The concentration 25 μM did not show any notable effect on fibroblast cell line but concentration 125 μM caused complete loss of live adherent cells. Compound 3 was more potent against HeLa cells, showing a weaker morphological effect at 25 μM but caused complete loss of live adherent cells at 125 μM, with only a weak effect on mitochondrial signal in fibroblast cells at 125 μM (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/full/10.1021/acsomega.5c01325#fig4">Figure 4</a>).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_22_23222_23234_Figure_4_Visualization_of_the_effects_of_compound_1_3_5_7_on_the_morphology_of_He_La_adenocarcinoma_cell_line_2d1b03cf08.jpg" alt="ACS Omega 2025, 10, 22, 23222–23234: Figure 4. Visualization of the effects of compound 1, 3, 5, 7 on the morphology of HeLa adenocarcinoma cell line (left) and primary human skin fibroblasts (right). DMSO was used as a solvent and as a control. Mitochondria are visualized using MitoTracker Red CMXRos (red), the actin cytoskeleton with Phalloidin (green), and nuclei with DAPI (blue)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_22_23222_23234_Figure_4_Visualization_of_the_effects_of_compound_1_3_5_7_on_the_morphology_of_He_La_adenocarcinoma_cell_line_2d1b03cf08.jpg 158w," sizes="100vw" width="1437" height="1421">Our study is also one of the few focusing on metabolites from smut fungi (specifically the order Exobasidiales), demonstrating that these overlooked fungi are potent producers of secondary metabolites. Little is known about the ecological role of the compounds they produce. The species we studied is not known to develop a pathogenic phase, which is otherwise typical for all members of the genus Exobasidium. Thus, it remains unclear whether these metabolites are involved in plant interactions. Instead, they might play a role in microbial competition within their environment.<h4>3. Experimental Procedures</h4><h5>3.1. General Experimental Procedures</h5>Optical rotations were determined using a Rudolph Research Analytical Autopol III automatic polarimeter in MeOH or acetone. NMR spectra were obtained using a Bruker Avance III 700 MHz spectrometer (700 MHz for 1H, 176 MHz for 13C) and a Bruker Avance III 600 MHz spectrometer (600 MHz for 1H, 151 MHz for 13C). All samples were measured in CD2Cl2. The spectra were referenced by the residual signal of the solvent (CD2Cl2: δH 5.323 ppm, δC 53.87 ppm). HRESIMS analysis and tandem mass spectrometry were performed on a Bruker qTOF Compact instrument with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II">Agilent 1290 Infinity II high-performance liquid chromatography (HPLC)</a> instrument equipped with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1514">Kinetex biphenyl columns</a> (100 × 2.1; 100 Å). Semipreparative HPLC was carried out on a Waters instrument equipped with a 2487 UV detector and Gemini C18 column (5 μm, 110 Å, 250 × 10.00 mm). Column chromatography (CC) was performed on Sephadex LH-20 (150 g, GE Healthcare Bio-Science, Sweden), STRATA C18 column (20 g, 55 μm, 70 Å Phenomenex); silica gel (0.035–0.070 mm, 70 Å, Lachner) fractions were analyzed by high-performance liquid chromatography (HPLC) with UV–vis detection using an Alliance HPLC 2988 dual photodiode array (PDA) detector equipped with a Gemini C18 analytical column (5 μm, 110 Å, 250 × 4.6 mm). The melting point was determined using CNYST micromelting point measuring instrument and are uncorrected.

Gunacins: Novel Benzo[g]chromene Derivatives from the Fungus Exobasidium sp. and Their Potent Anti-Leishmania and Trypanosoma Activities

News from LabRulezLCMS Library - Week 52, 2025

News from LabRulezLCMS Library - Week 02, 2026

Bacillus species produce diverse bioactive lipopeptides that contribute to their ecological and biocontrol functions, yet some families remain poorly characterized. This study focuses on kurstakins, a lipopeptide family associated with biocontrol activity, using metabolomic, structural, and multiomics approaches.Metabolomic analysis of a Bacillus cereus extract revealed approximately 50 kurstakin analogs with diverse linear and branched lipid tails. Two new analogs were isolated and fully characterized by NMR, providing the first experimental determination of absolute configuration within this family. Integration of public genomic and MS data further demonstrated the broad chemical diversity and global distribution of kurstakins, highlighting their potential ecological significance in Bacillus-based biocontrol systems.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212">Structural Characterization and Multiomics Analysis Reveal Extensive Diversity and Global Distribution of Kurstakin Lipopeptides</a></h4>Rose Campbell, and Emily Mevers*J. Nat. Prod. 2025, 88, 12, 2960–2967<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.jnatprod.5c01212">https://doi.org/10.1021/acs.jnatprod.5c01212</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Lipopeptides are a well-known family of microbial natural products that possess a range of applications, including their potential medicinal uses and as crop biocontrol agents. <a href="javascript:void(0);">(1)</a> Several lipopeptides or synthetic analogs are FDA-approved for the treatment of infectious diseases, including as antibiotics (e.g., polymyxin B, oritavancin, telavancin, and daptomycin) and antifungals (e.g., rezafungin and micafungin), with many others in various stages of clinical trials. <a href="javascript:void(0);">(2,3)</a> Their bioactive properties arise from their amphiphilic structures, which consist of a fatty acid tail (lipid) and a polar peptide. The lipopeptide antibiotics are known to bind to cell membranes, forming pores that lead to depolarization. <a href="javascript:void(0);">(4)</a> Lipopeptides are commonly produced by a diverse range of microorganisms, including bacteria, fungi, and cyanobacteria; however, Bacilli are particularly well-known for their ability to produce structurally diverse bioactive lipopeptides. This includes the widespread production of surfactins, iturins, fengycins, polypeptins, and octapeptins. <a href="javascript:void(0);">(2,5)</a> This biosynthetic capability has led to their wide use as biocontrol agents in agricultural settings, such as Bacillus thuringiensis being leveraged for insect and disease control and Bacillus velezensis for controlling plant pathogens. <a href="javascript:void(0);">(6,7)</a> The diverse lipopeptides produced by these strains have been shown to work synergistically as antifungals, biofilm formation regulators, and plant defense modulators. <a href="javascript:void(0);">(5,8)</a>Although many lipopeptides have been well characterized from Bacilli, this does not represent their full biosynthetic potential. <a href="javascript:void(0);">(9)</a> In addition, many lipopeptide families have been mentioned in the literature, but lack appropriate analytical data to confirm their planar and secondary structures and the diversity of the family. <a href="javascript:void(0);">(2)</a> This includes the kurstakins, gavaserin, and saltavalin, which have all been implicated to play a critical role in the biocontrol properties of the producing organisms. <a href="javascript:void(0);">(10)</a> The kurstakins were first discovered to be produced by B. thuringiensis, and have been considered a biomarker for Bacillus cereus group species. <a href="javascript:void(0);">(11,12)</a> However, they have since been isolated from a range of bacteria, including non-cereus group Bacillus species (e.g., B. subtilis, B. licheniformis) <a href="javascript:void(0);">(13,14)</a> and more distantly related bacteria (e.g., Enterobacter cloacae, and Citrobacter sp.). <a href="javascript:void(0);">(15,16)</a> Several of the producing strains have been studied extensively for their potential use as a biocontrol agent, due to their antimicrobial activity and ability to swarm and produce protective biofilms, <a href="javascript:void(0);">(12,17,18)</a> which has been attributed in part to the kurstakins. <a href="javascript:void(0);">(19−22)</a> Four cyclic analogs, kurstakins 1–4 (1–4), were elucidated through tandem mass spectrometry (MSMS) analyses and chemical derivatization. <a href="javascript:void(0);">(12)</a> There have been reports of many additional analogs, but these reports are primarily based on MS analyses. <a href="javascript:void(0);">(23−25)</a> The studies indicate that all analogs contain the same peptide core, but incorporate diverse lipid tails. Additionally, amino acid configurations were deduced through bioinformatic analyses alone, <a href="javascript:void(0);">(17)</a> which can only predict the configuration of the α-position. Therefore, chemical analyses are needed to unequivocally determine the amino acid configurations, particularly of the β-hydroxyl in the threonine residue. Herein, we leveraged our metabolomic data set from marine bacteria to characterize the kurstakins, including the first NMR analysis, report of two new analogs, kurstakins 5 and 6 (5–6), deduction of the absolute configuration, and analysis of the true chemical diversity and distribution of the kurstakins.<h4>Results and Discussion</h4>Analysis of a Global Natural Product Social (GNPS) molecular network representing the chemical potential of diverse marine bacteria from our internal library revealed that it contained a rather large spectral family, with masses ranging from 850 to 940 m/z (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig1">Figure 1</a>A). <a href="javascript:void(0);">(26)</a> A subset of these nodes were identified as 1–4 based on their exact mass and key fragment ions (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig1">Figure 1</a>B). A peak at 110.1 m/z, which putatively represents a decarboxylated histidine, was abundant in all MSMS spectra and became a key identifying fragment (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#notes-2">Figure S1</a>). Other nodes matched masses of reported kurstakin analogs with hypothetical structures, <a href="javascript:void(0);">(15,19,23,27)</a> as well as new analogs. Based on key MS fragments, this spectral family represents both cyclic and linear core peptide sequences that have incorporated various lipid tails (e.g., C9–C14 with and without hydroxylation; <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#notes-2">Figure S2–S3</a>).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Nat_Prod_2025_88_12_2960_2967_Figure_1_A_Molecular_family_of_kurstakins_found_in_the_marine_isolates_library_8818fa75c4.jpg" alt="J. Nat. Prod. 2025, 88, 12, 2960–2967: Figure 1. (A) Molecular family of kurstakins found in the marine isolates library, showing structural diversity in tail structure and peptide linearity. (B) Key common fragments of each core peptide used to identify the kurstakins. Fragments not including histidine were rarely observed. (C) Overlaid EICs of all kurstakin masses observed in an enriched semipure fraction, 850–996 m/z, representing a large diversity of cyclic (aqua), linear (blue), and hydroxyl-tail (red) analogs." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Nat_Prod_2025_88_12_2960_2967_Figure_1_A_Molecular_family_of_kurstakins_found_in_the_marine_isolates_library_8818fa75c4.jpg 245w," sizes="100vw" width="1600" height="889">To characterize the structural breadth of the kurstakin family, the producing strain, B. cereus EM195W, was grown in large-scale (8 L) liquid media containing hydrophobic resins (HP-20, XAD4, and XAD7) to capture the secreted small molecules. After 9 days, the resin was extracted with organic solvents to generate a crude extract. The crude extract was first subjected to reverse-phase (RP) flash chromatography to yield eight refined fractions. MS analysis revealed that the kurstakins were present in the nonpolar fractions, which were combined and processed through normal-phase (NP) flash chromatography, generating an additional 13 fractions. Fractions that were highly enriched with the kurstakins were reanalyzed by high-resolution liquid chromatography-MS (HR-LCMS), revealing the presence of many more analogs than previously detected in the original GNPS analysis. Manual interpretation of the MS data indicated that there is significantly more tail variation than previously thought, which was likely overlooked due to the isobaric nature of many of the analogs and low relative abundance of several analogs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig1">Figure 1</a>C). In total, 31 masses ranging from 850–996 m/z were detected, representing approximately 50 distinct analogs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#notes-2">Table S1, Figure S4</a>). The breadth of these analogs spanned tail lengths ranging from C9–C17, most of which appeared as all possible combinations of linear- or cyclic-peptide core with and without hydroxylated tails, and represented many previously undescribed compounds.To discover the breadth of tail diversity, we turned to gas chromatography-MS (GC-MS) analysis. A portion of one of the semipurified NP fractions was treated with HCl (aq) to liberate the free acid, which was subsequently methylated with TMS-diazomethane. The methylated fatty acids were analyzed by GC-MS, comparing the retention times and fragmentation patterns with authentic standards of linear, iso-branched, and anteiso-branched fatty acid methyl esters (LCFA, iBCFA, and aBCFA, respectively). <a href="javascript:void(0);">(34)</a> Comparing the derivatized natural material to the LCFA standards revealed the presence of linear fatty acid tails ranging in length from C9–C18 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jnatprod.5c01212#fig2">Figure 2</a>A). This agreed with the masses found on the HR-LCMS, but with the addition of the C18 LCFA.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Nat_Prod_2025_88_12_2960_2967_Figure_2_Chemical_characterization_of_the_family_of_kurstakins_405ad6222e.jpg" alt="J. Nat. Prod. 2025, 88, 12, 2960–2967: Figure 2. Chemical characterization of the family of kurstakins. (A) GC-MS traces (EIC of common 74.1 m/z fragment) of the semipure B. cereus EM195W fraction (purple) compared to the LCFA (blue) and iBCFA (aqua) standards. (B) Summary of all fatty acid tails identified in kurstakins produced by EM195W." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Nat_Prod_2025_88_12_2960_2967_Figure_2_Chemical_characterization_of_the_family_of_kurstakins_405ad6222e.jpg 245w," sizes="100vw" width="1300" height="673"><h4>Experimental Section</h4><h5>General Experimental Procedures</h5>Optical rotation data were recorded on a JASCO P-2000 polarimeter. NMR spectra were recorded in d6-DMSO with the residual solvent peak as an internal standard (δC 39.5, δH 2.50) on a Bruker Avance III 600 MHz instrument equipped with a triple resonance inverse (CP-TCI) Prodigy N2 cooled CryoProbe (1H 600 MHz; 13C 150 MHz) or a Bruker Avance II 500 MHz instrument equipped with a CPBBO Prodigy N2 cooled CryoProbe (1H 500 MHz; 13C 125 MHz). TFA fuming was monitored on a JEOL ECZ 400 MHz instrument equipped with a Royal HFX probe (1H 400 MHz). GC-MS data were obtained using an <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=5975C">Agilent 5975C VL MSD GC-MS</a>. LR-LCMS data were obtained using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1200">Agilent 1200 series HPLC system</a> equipped with a photodiode array detector and a Thermo LTQ mass spectrometer. HR-ESIMS and HR-MSMS were carried out using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6530">Agilent 6530 Q-TOF</a> equipped with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II">1290 Infinity II UPLC system</a> or a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/403">Thermo Orbitrap Exploris 120 mass spectrometer</a>. Reverse-phase flash chromatography purification was carried out with a Biotage Selekt automated flash chromatography system. HPLC purifications were carried out using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+Infinity+II">1260 Infinity II HPLC system</a> equipped with a photodiode array detector. All solvents were of HPLC quality.

Structural Characterization and Multiomics Analysis Reveal Extensive Diversity and Global Distribution of Kurstakin Lipopeptides

Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 12th January 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!<blockquote>👉 <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library">SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</a></blockquote><blockquote>👉 Need info about different analytical techniques? Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library">LabRulezGCMS</a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library">LabRulezICPMS</a> libraries.</blockquote>This week we bring you application notes by Agilent Technologies, Shimadzu, Waters Corporation and poster by Thermo Fisher Scientific / HPLC!<h3>1. Agilent Technologies: B Vitamin Analysis in Dietary Supplements on the Agilent 1290 Infinity III LC System</h3>Using ion-pairing reagent and supported by time‑saving automation of routine tasks using the Agilent InfinityLab Assist<ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_b_vitamin_dietary_supplements_1290_infinityiii_lc_5994_8826en_agilent_56b5eb2fb1.pdf">Full PDF for download</a></li></ul>There is a huge variety of (multi)vitamin supplements on the market, with different vitamin combinations and different concentrations available. To ensure that the claimed vitamin contents are met, analysis of those supplements is&nbsp;necessary.&nbsp;To ensure constant and good analytical performance, preparation of the analytical system is fundamental. There are different approaches to preparing the system for analysis. Purging the solvent lines from bottle to pump removes possible air bubbles, especially after longer breaks between instrument usage or changing eluent to the ion-pairing and buffer-containing eluent after the weekend. Equilibration can either be done by a calculated time (10–20 column volumes)1 , set as a fixed experimentally determined value, or manually monitored. Using a fixed time for equilibration can lead to unnecessary high solvent consumption, as equilibration may already be established and no monitoring was previously done. In contrast, manual monitoring of equilibration can be time-consuming and error-prone. For example, if there are no written rules regarding key points for equilibration of the column, its determination can be subjective (constant&nbsp;pressure and baseline of e.g. UV absorption).&nbsp;As vitamins are grouped according to their water solubility and not their chemical structure, separation behavior can greatly differ as they have different charges at physiological pH. In such cases, ion-pairing reagents such as hexane sulfonic acid can enable the separation of those vitamins while using a reversed phase column. As ion-pairing LC depends on good equilibration to ensure the presence of ion‑pairing reagent within the system, the process of preparing the instrument is an inevitable step. This is essential for good and reproducible analytical results.&nbsp;With the Agilent InfinityLab Assist, making your instrument ready is no longer a time-consuming and unpleasant procedure.1 This application note demonstrates the separation of multiple B vitamins in multivitamin tablets and effervescent tablets using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=1290+Infinity+III">Agilent 1290 Infinity III LC System</a>. Furthermore, it is shown how the InfinityLab Assist can help users ensure good chromatographic results, save time, and reduce errors by replacing manual tasks with InfinityLab Assist tasks.&nbsp;<h4>Experimental&nbsp;</h4><h5>Equipment&nbsp;</h5><ul><li>Agilent 1290 Infinity III High-Speed Pump (G7120A)&nbsp;</li><li>Agilent 1290 Infinity III Multisampler (G7167B) with Agilent InfinityLab Sample Thermostat (G4761A)&nbsp;</li><li>Agilent 1290 Infinity III Multicolumn Thermostat (G7116B) with Agilent InfinityLab Quick Connect Heat Exchanger (G7116-60015)&nbsp;</li><li>Agilent InfinityLab Level Sensing (G7175A)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+Assist">Agilent InfinityLab Assist</a> (G7180A) with Agilent InfinityLab Assist Control Software, version 2.0&nbsp;</li><li>Agilent 1290 Infinity III Diode Array Detector (G7117B) with Agilent InfinityLab Max-Light Cartridge Cell (10 mm, 1.0 µL, G4212-60008)&nbsp;</li></ul><h5>Software&nbsp;</h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=OpenLab+CDS">Agilent OpenLab CDS,</a> version 2.8, update 7 (or later versions)&nbsp;</li></ul><h5>Column&nbsp;</h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+Poroshell+120+EC-C18">Agilent InfinityLab Poroshell 120 EC-C18</a>, 2.1&nbsp;mm × 100 mm, 2.7 µm column (p/n 695775-902).</li></ul><h4>Results and discussion&nbsp;</h4><h5>Separation of vitamin B1, B2, B5, B9, and B12&nbsp;</h5>To analyze vitamin B1, B2, B5, B6, B7, B9, and B12 in vitamin supplements, different columns, mobile phases, and gradients were evaluated. The selected chromatographic conditions (Table 1) showed the best separation from peak to peak and additional matrix peaks. Hexane sulfonate was needed as an ion-pairing reagent to ensure good peak shapes, as all vitamins investigated, except vitamin B12, are expected to be charged based on their pKa values.3 The method detects the B vitamins investigated in vitamin tablets and effervescent tablets as demonstrated in Figure 1, showing chromatograms of aqueous standards and real samples. As some vitamins are present at higher doses, such as the 12 mg tablet (vitamin&nbsp;B5) compared to the 2.5 µg tablet (B12) in supplement 3 (Figure 1), parallel analysis is rather difficult, and not all vitamins present were detectable in each&nbsp;supplement.<h4>Conclusion&nbsp;</h4>Using the Agilent 1290 Infinity III LC System setup detection of vitamins B1, B2, B5, B6, B7, B9, and B12 in effervescent tablets and multivitamin supplement matrix, good separation was achieved. Using the Agilent InfinityLab Assist "Make&nbsp;Ready" task, the system was prepared for B vitamin analysis using only one automated task and no manual monitoring. Running this task automatically by scheduling saved more than 20 minutes of working time. Additionally, the time required for medium equilibration monitoring sensitivity was reduced by approximately six minutes, compared to manual equilibration. Furthermore, using the schedulable InfinityLab Assist "Standby" task, the risk of errors regarding instrument standby can be drastically reduced. User-defined settings enable adjustment for different purposes, such as switching of all modules except the sampler thermostat.2 The ability to remove the used buffer and ion-pairing reagent from the flow path for longer storage by scheduling a standby task also greatly helps to prevent salt precipitation in the system without additional effort. Also, the InfinityLab Assist can clearly help to accelerate checking the instrument status while preparing the instrument, as well as during running&nbsp;samples.2<h3>2. Shimadzu: Analysis of Ammonia as an Impurity in Fuel Cell Grade Hydrogen According to ISO 14687 Grade D Using a Gas Generator</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_01070_en_43a535bf87.pdf">Full PDF for download</a></li></ul>Hydrogen is expected to be a new power and heat source to reduce greenhouse gas emissions. Hydrogen is an easily available substance that can be produced from the electrolysis of water in our daily lives. However, when used in fuel cells, it is necessary to control impurities in hydrogen gas because certain impurities introduced during the manufacturing process deteriorate the electrode and electrolyte membrane, reducing the performance of the fuel cell or shortening its life. When ammonia is used as a carrier of hydrogen gas due to its distribution as a chemical, there is a possibility that ammonia may be introduced into hydrogen. This article presents an example of analysis of ammonia in hydrogen using an ion chromatograph (IC) based on the standard values described in ISO 14687 Grade D.1)<h4>Analysis of Ammonium Ion by IC</h4>A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=HIC-NS">non-suppressor IC (HIC-NS)</a> was used to analyze NH4. Since the IC can selectively measure ions dissolved in the sample, it is less affected by contaminants than other methods and can measure NH4 with high accuracy. In addition, by using the same device together with a suppressor IC, it is possible to measure inorganic halogens and formic acid, which are other impurities listed in ISO 14687. Table 1 shows the analytical conditions for NH4, Fig. 2 shows the chromatogram of a standard sample at a concentration near the specified value (25 μg/L), and Fig. 3 shows the calibration curve in the range from 2.5 μg/L to 200 μg/L. As shown in Fig. 3, the linearity was greater than 0.9999. Table 2 shows the reproducibility of the sample at 10 μg/L, which is around 1/2 of the specified concentration, repeated 7 times.<h4>Conclusion</h4>Ammonia, an impurity in hydrogen, was analyzed using a nonsuppressor IC, and it was confirmed that the method met the standard value specified by ISO 14687 Grade D. In addition, when ammonia standard gas near the standard value was mixed into hydrogen using a gas generator, a good recovery rate was obtained, which confirmed the validity of the method including the collection flow of pretreatment.<h3>3. Thermo Fisher Scientific / HPLC: High-sensitive multi-attribute analysis of ADCs under native conditions by using an online multiple heart-cut 2D-LC-HRAM mass spectrometry system</h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_high_sensitive_multi_attribute_analysis_adcs_native_conditions_po_en_28e7e0b002.pdf">Full PDF for download</a></li></ul>ADCs are a rapidly expanding class of biotherapeutics for cancer treatment. Critical quality attributes such as charge heterogeneity, DAR, and drug distribution are essential for ensuring the safety, stability, and efficacy of ADCs. Strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) are conventional methods employed for charge variant analysis and DAR determination of ADCs1. However, these methods frequently utilize MS-incompatible salts, limiting their use for MS characterization. Online 2D-LC addresses this by enabling desalting in the second dimension(2D) and offering multi-attribute analysis in one method. Here, multiple heart-cut 2D-LC-HRAM MS methods were developed to achieve high sensitivity for the multi-attribute analysis of polatuzumab vedotin under native conditions.<h4>Methods</h4>A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/742">Thermo Scientific Vanquish 2D UHPLC system</a>, coupled with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/402">Thermo Scientific Orbitrap Exploris 240 mass spectrometer</a> with Biopharma option, were utilized to configure the multiple heart-cut 2D-LC-HRAM MS system. HIC-SEC and SCXSEC methods were developed and optimized for the charge variants, drug-to-antibody ratio (DAR) and drug load distribution (DLD) characterization of ADCs under native conditions.<h4>Conclusions&nbsp;</h4>In this study, an online multiple heart-cut 2D-LC-HRAM MS system was configured and optimized for the multi-attribute analysis of polatuzumab vedotin. This approach provides detailed and comprehensive insights into the charge heterogeneity analysis, DAR, and DLD analysis of polatuzumab vedotin under native conditions.&nbsp;<ul><li>The online multiple heart-cut 2D-LC system enables the direct transfer of SCX and HIC fractions to mass spectrometry analysis by utilizing SEC as a desalting step, thereby overcoming the MS-incompatibility buffer in the LC separation.&nbsp;</li><li>The backflush transfer of fractions was employed to enhance peak shape and sensitivity in the 2D SEC separation.&nbsp;</li><li>The 2D SEC-MS analysis enables the characterization of ADCs under native conditions.&nbsp;</li><li>The high sensitivity of the Orbitrap Exploris 240 MS allows for the identification of fractions with low abundance in the 1D chromatography</li></ul><h3>4. Waters Corporation: Automated Multiple Reaction Monitoring (MRM) Method Development for Peptide Drugs Using waters_connect for Quantitation Software</h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009123_en_1f51e50be8.pdf">Full PDF for download</a></li></ul><h5>Benefits&nbsp;</h5><ul><li>Rapid evaluation and optimization of MRM conditions freeing up bioanalysts to perform other valuable tasks</li><li>Interactive graphical review of results facilitating informed decision making.&nbsp;</li><li>Elimination of transcription errors via direct transfer of MRM results to the LC-MS method editor.</li></ul>LC-MS/MS is the technology of choice for the quantification of drugs, metabolites, and biomarkers in biofluids, typically employing tandem quadrupole mass spectrometry (MS) with detection via MRM. MRM method development for classic small molecule drugs involves the selection of the best precursor and product ion followed by optimization of cone voltages and collision energies. This is achieved by infusion or LC injection of the analyte(s) of interest. For small molecule drug compounds, this process is usually straightforward and lends itself to automation as they largely generate singly charged ions. However, in electrospray mass spectrometry, biomolecules such as peptides, oligonucleotides, and proteins normally form several multiply charged precursor ions, e.g., M+3H3+, M+4H4+. This significantly complicates MRM method development, as each charge state can generate multiple fragment ions, all of which need to be compared both within and across each charge state to choose the best transition or transitions for analyte measurement.&nbsp;The MRM Optimization tool with the waters_connect for Quantification Software has been specifically designed to address the challenge of MRM method development for multiply charged biomolecules. The Optimization tool facilitates the automatic acquisition of data from multiple precursor and product ion and collision energy combinations across several charge states, with the results reviewed via an interactive graphical interface. Here, the use of this optimization is illustrated, using the GLP-1 peptide, semaglutide, as an example.<h4>Experimental</h4><h5>Mass Spectrometry</h5>Mass spectrometry was performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2100">Waters Xevo™ TQ Absolute XR Mass Spectrometer</a>, operated in positive ion electrospray ionization mode (ESI). Nitrogen was used as the nebulizer gas and argon as the collision gas. The cone voltage, collision energy, and MS/MS transitions were adjusted and evaluated using the waters_connect MRM Optimization tool.<h5>Informatics&nbsp;</h5>The mass spectrometer was controlled and resulting data evaluated using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1544">waters_connect for Quantitation</a> Application Manager version 1.9.<h4>Conclusion&nbsp;</h4>The number of candidate biomolecule drugs, peptides, oligonucleotides, and proteins in pharmaceutical discovery and development is steadily increasing. These biomolecules require the development of quantitative LC-MS/MS assays to support discovery, preclinical, and human DMPK studies. Unlike small molecules, these biomolecules form multiply charged ions in the MS source, making MRM method development more challenging, as multiple charge state–product ion combinations must be evaluated and compared. The Optimization tool in waters_connect for Quantitation Software provides an automated, fast platform for the optimization and evaluation of the precursor ion → product ion pairs formed at each charge state, simplifying the process of LCMS/MS method development for biomolecules.

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Structural Characterization and Multiomics Analysis Reveal Extensive Diversity and Global Distribution of Kurstakin Lipopeptides

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