The dried roots of Stemona tuberosa, known as "Bai-Bu" in traditional Chinese medicine, have been employed as a therapeutic agent for the treatment of pertussis and tuberculosis. The chemical composition of S. tuberosa is dominated by alkaloids which have been reported to possess antifibrosis effect. Herein, ELISA kit and immunofluorescence analyses showed that the alkaloid of stemoninoamide (Stem) isolated from S. tuberosa exhibited inhibitory effect on the production of α-SMA and Col-1 of HFL1 cells stimulated by TGF-β1. Further lipidomic analysis based on LC-MS/MS revealed differential metabolites via multivariate statistical analysis. Pathway enrichment analyses indicated that differential metabolites were mostly implicated in S1P/S1PR signaling pathway, which could be potentially associated with the antifibrosis mechanism of Stem.
An improved method was proposed, which addressed the technical challenge of incomplete combustion and conversion of high-temperature, high-pressure metamorphic graphite-a chemically stable material for conventional EA-IRMS method analysis. The method was based on the addition of an efficient oxidant. Using graphite from a Sri Lankan charnockite (G1) and graphite containing pyrite (G2) as test materials, the effects of oxygen injection modes and solid oxidants (CuO and V2O5) on combustion efficiency were evaluated. It was demonstrated that merely increasing oxygen flow rate or duration failed to achieve complete oxidation of the samples, whereas the addition of V2O5 effectively resolved the problem. By optimizing reaction conditions, an optimal analysis method was proposed: mixing the graphite sample with V2O5 at a mass ratio of no less than 1:6 and injecting oxygen at a flow rate of 175 mL/min for 6 s. Under these conditions, both types of refractory graphite samples achieved complete combustion, significantly enhancing carbon conversion efficiency, ensuring the accuracy of δ13C analyses. The method substantially extends the applicability of EA-IRMS technology for isotope analysis of inert geological carbon materials, providing reliable and critical support for research on deep carbon cycling, graphite deposit genesis, and related studies.
Computational chemistry has played a key role in helping scientists understand ionization and molecular dissociation in the gas phase, which has contributed to its growing popularity among users of various mass spectrometry techniques. Early studies laid a strong foundation for applying computational methods to explore the stability and reactivity of gas-phase ions, making these approaches more accessible and widely used across different research areas. In this work, we present a practical guide for effectively using computational chemistry in mass spectrometry studies. By comparing experimental results obtained in ESI-MS/MS with those from quantum chemical models (DFT and composite methods) and spectra simulations (QCxMS and CFM-ID), we aim to provide researchers with clear steps for studying organic molecules from ESI-MS and MS/MS data and understanding the protonation in mass spectrometry. Proton affinity and gas-phase basicities were calculated and the protonation sites were proposed. These results were compared to atomic charges, molecular orbitals, and Quantum Theory of Atoms-in-Molecules (QTAIM) calculations. Fragmentation mechanisms were proposed from protonated species, and the MS/MS spectra were compared to those predicted using CFM-ID and QCxMS methods. The 2-methyl-1,4-naphthoquinone analysis was used as a reference molecule for computational studies and benchmark. It is suggested the useful toolkits to describe molecular ionization/dissociation in ESI-MS and ESI-MS/MS studies from quantum chemical calculations by using a guide for the systematic application of computational chemistry in mass spectrometry and fragmentation mechanisms studies. Finally, an application was demonstrated for 2-hydroxy-3-benzamine-1,4-naphthoquinone ionized in positive mode, highlighting the versatility of this guideline for studies involving more structural complexity.
Olive oil is widely consumed for its nutritional and health benefits, yet its high market value makes it vulnerable to adulteration and mislabeling. Current analytical approaches for assessing olive oil quality and authenticity rely largely on indirect analyses, such as fatty acid methyl ester (FAME) profiles, peroxide values, or UV absorbance, and do not account for olive oil's triglycerides content. In this manuscript, we demonstrate the application of gas chromatography-mass spectrometry (GC-MS) with cold electron ionization (Cold EI) for the direct analysis of olive oil based on its glyceride profiling. Cold EI employs a supersonic molecular beam interface with a contact-free fly-through ion source, providing vibrational cooling that enhances the abundance of molecular ions. This enables direct GC-MS analysis of all glycerides and acids without derivatization, with total analysis time of 19 min. We analyzed multiple commercial olive oil brands, certified reference material, and intentionally adulterated samples containing canola and/or sunflower oil. The resulting mass chromatograms and triglyceride molecular ion patterns allowed clear discrimination between pure olive oil, adulterated oils, and degraded (oxidized) samples. Adulteration at the 30% level was detected through distinct hybrid triglyceride patterns, although aging effects were uniquely identified by increased diglyceride and free fatty acid signals. High sensitivity was demonstrated by the confident detection of olive oil at trace levels down to 100 pg on-column using selective ion monitoring. These results establish GC-MS with Cold EI as a rapid, sensitive, and highly informative technique for ensuring the authenticity, quality, and consumer safety of olive oil.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of saccharides, particularly high-molecular-weight polysaccharides, is often hampered by low ionization efficiency. To overcome this limitation, we developed a novel composite matrix comprising 4-hydrazinoquinazoline (4-HQ) and caffeic acid (CA). This matrix synergistically enhances saccharide detection: 4-HQ specifically targets the hemiacetal termini of reducing saccharides via hydrazone formation, thereby improving ionization efficiency, while CA maintains an acidic condition for derivatization and extends the detectable mass range. Using dextran 6 k as a model, we systematically optimized the 4-HQ/CA ratio, reaction temperature, and reaction time. The optimized 4-HQ/CA matrix enabled direct profiling of oligo/polysaccharides in inulin and garlic extracts, as well as N-glycans from ovalbumin and fetuin. Notably, even for non-reducing saccharides where derivatization is precluded, 4-HQ/CA outperformed conventional 2,5-dihydroxybenzoic acid (DHB) by detecting more species with higher signal-to-noise ratios. This versatile composite matrix shows potential for robust analysis of saccharides in glycobiology and food science.
Gas chromatography-mass spectrometry (GC-MS) is the reference method for urine organic acid analysis but requires complex sample preparation and derivatization, limiting routine clinical use. We developed and validated a targeted Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying urinary organic acids relevant to the evaluation of inborn errors of metabolism. The assay demonstrated excellent linearity, precision, accuracy, analytical sensitivity, and good agreement with GC-MS. Organic acidurias and other metabolic disorders were accurately identified through characteristic metabolite elevations. The simplified workflow eliminates derivatization and enables rapid, high-throughput implementation for newborn screening confirmation, metabolic screening, and patient monitoring in pediatric clinical laboratories.
Poor diet is now the leading cause of early death globally. In part, this is because our complex food supply chains are increasingly at risk of overprocessing, contamination, low nutrient content, and economically motivated fraud. Chemical testing can offer insights into these concerns, but testing methods are frequently impractical. Extra virgin olive oil (EVOO) is a premium food of high nutritional value, but because of its growing popularity and high price, it can be a target for mislabeling, substitution, dilution, and/or false claims of origin. Rapid and accurate testing methods for its characterization are therefore increasingly important. We used two direct forms of mass spectrometry (MS)-laser desorption/ionization (LDI) MS and direct analysis in real time (DART) MS-to obtain complex chemical signatures of edible oils. The data generated on a set of reference samples were then used to develop and train three independent machine learning (ML) models that assess key characteristics of a test oil. We also developed a proof-of-concept DART-MS/MS assay add-on for the quantification of bioactive phenols in EVOO. Our approach accurately predicts several attributes of an edible oil based on novel markers and intricate patterns within the acquired data. Further, pure reference standards and an isotopically-labeled internal standard allow accurate quantification of the constituent phenols. Because there is no chromatography, both the mass fingerprints and quantification can be performed in seconds-minutes. The method uses low (milliliter) volumes of sample and green solvents, and when combined with ML, it offers rapid data analysis and comprehensive result interpretation.
Insulin aggregation and oligomerization present significant challenges in both therapeutic formulation and fundamental studies of amyloid formation, particularly due to the transient and heterogeneous nature of early-stage oligomers. Here, we employ ion mobility-mass spectrometry (IM-MS) to characterize the oligomeric distributions of human insulin and two clinically relevant analogs, aspart (rapid-acting) and glargine (long-acting), in excipient-containing solutions designed to stabilize native forms of insulin and under aggregation-inducing conditions. Comparison of insulin analogs revealed distinct aggregation propensities that correlate with each analog's therapeutic design. In general, stable zinc-coordinated hexamers formed most readily in the presence of excipients, and various oligomers formed under aggregation-inducing conditions, with a noticeable prevalence of heptamer formation. We further investigated the structure and stability of native hexamers and non-native heptamers using collision-induced dissociation and collision-induced unfolding experiments. Although the expected single-monomer ejection was the main dissociation pathway for both species, zinc-coordinated hexamers also dissociated into two zinc-adducted trimers, whereas heptamers dissociated into dimer/pentamer and trimer/tetramer pairs. Gas-phase unfolding indicated conservation of subunit tertiary structure in hexamers and no distinct folded structures in heptamer subunits. Overall, this work demonstrates the utility of IM-MS as a rapid, high-resolution platform for probing insulin aggregation pathways and evaluating current and future insulin analog formulations.
Remnants of erythrocyte removal from blood constitute therapeutic plasma-a water-based solution containing mainly platelets, extracellular vesicles (EVs), protein and lipid complexes and ions. Plasma-based therapies, including their use to accelerate wound healing in human and animal patients, are increasingly gaining prominence in regenerative medicine; however, methods for the optimal preparation of individual plasma are still under development. Rapid and accurate analysis of plasma composition is essential for the development of therapeutic applications and for ensuring the quality of plasma products before patient administration. This study of equine plasma presents a novel approach that utilises a newly developed automated well-plate sampling system using laser-assisted rapid evaporative ionisation mass spectrometry (LA-REIMS) for rapid plasma profiling under varying sample preparation conditions. This platform provided comprehensive lipidomic and metabolic analyses that gave insights into the molecular composition of plasma within seconds and without extensive sample preparation. The results indicated that specific phosphatidylcholines (PC(34:2), PC(36:2), PC(36:3)) and fatty acids were sensitive to centrifugation parameters, suggesting their potential as indicators of plasma stability and quality. The results were compared with the platelet and EV content of the plasma. The number density (n) of platelets and cellular fragments (P2 particles) was determined by flow cytometry (FCM), whereas EVs were characterised by interferometric light microscopy (ILM). The samples were imaged by scanning electron microscopy (SEM). A clear correlation was observed between n of P2 particles (but not of EVs) and lipid profiles, suggesting platelets' critical role in plasma composition. These findings emphasise the potential of the LA-REIMS platform for high-throughput, rapid plasma analysis, quality control and diagnostics. The system's ability to quickly assess plasma quality highlights its practical use in validating plasma prior to therapeutic applications.
Organotin (IV) compounds are known to induce apoptosis via the intrinsic mitochondrial pathway, which is a key mechanism of effective anticancer therapy. Their ability to selectively promote apoptotic cell death highlights their potential as chemotherapeutic agents. In this study, the in vitro effects of two triorganotin compounds, tributyltin propionate and tributyltin salicylate, on the human breast cancer cell line MDA-MB-231 were evaluated. In addition to their proven antitumor activity, these compounds may act as synthetic ligands for nuclear retinoid X receptors. Protein expression profiles were examined using gel electrophoresis and MALDI-TOF mass spectrometry, with a particular focus on heat shock proteins (HSPs), which are commonly overexpressed in cancer cells and contribute to tumor progression and therapeutic resistance. Both triorganotin derivatives significantly reduced HSP expression, suggesting that HSPs could be a promising target in cancer therapy.
Peptides are increasingly employed as active ingredients in both therapeutic and cosmeceutical applications due to their high biological specificity, favorable safety profiles, and expanding market relevance. In topical formulations, peptide activity is typically confined to the skin, where widespread dermal proteases may significantly affect their stability and efficacy. Despite the growing use of bioactive peptides in dermatological and cosmeceutical products, robust analytical methodologies for assessing their susceptibility to dermal enzymatic degradation remain limited. In this study, a two-dimensional HPLC-MS/MS (2D-HPLC-MS/MS) method based on ion trap detection was developed and validated for the quantitative evaluation of peptide stability in human skin homogenate (HSH). The analytical setup integrates online cleanup with chromatographic separation on a silica-based pentafluorophenyl (PFP) column, enabling reliable analysis of peptides with different polarity profiles within a single workflow. The system enables direct injection of sample solutions with relatively high organic solvent content, allowing limited sample dilution and preservation of analytical sensitivity. A key feature of the method is the use of isomeric peptide analogues as internal standards, monitored under identical MS/MS conditions as their corresponding analytes. This approach provides effective correction for ionization and fragmentation variability while offering a practical alternative to stable isotope-labelled standards. The ion trap mass analyzer ensured controlled and reproducible fragmentation behavior, supporting robust quantitative performance. The developed method demonstrated satisfactory linearity, sensitivity, precision, accuracy, and minimal matrix effects. Application to stability studies of three peptides with distinct polarity profiles confirmed its suitability for monitoring degradation kinetics in HSH. Overall, this 2D-HPLC-MS/MS strategy provides a versatile analytical platform for dermal peptide stability assessment and supports early-stage screening and preclinical studies of bioactive peptides intended for topical applications.
The interaction between solvent, solute and physical phenomena (e.g., photon-emitting biomolecules) constitute one of the most fundamental, yet challenging, frontiers in modern biophysics and analytical chemistry. However, standard mass spectrometry instrumentation is often insufficient to yield useful data in this domain due to the solvent molecule bind alteration induced by high electric field presents in the usually employed ionization sources (e.g., Electrospray and Atmospheric Pressure Chemical Ionization). The challenge is to transport analyte ions from the condensed liquid phase into the high-vacuum gas phase of the mass analyzer with minimal voltages-induced loss of supramolecular structure or structural scrambling while preserving the solvent environment structure. In this work, we employed no-discharge ND-APCI that has been rigorously validated in regulatory environments operating in no voltage ionization conditions to investigate the interaction of Albumin and water molecule environment under the irradiation of photon emitted by means of quantum dots technology at different simultaneous wave lights (800 nm, 525 nm, 445 nm). The obtained results, in terms of albumin-solvent (water) interaction, are consistent with a protein-dependent change in the proton affinity of albumin and are corroborated by complementary pH and colorimetric (Coomassie) measurements; their possible mechanistic origin is discussed.
Dehydroepiandrosterone sulfate (DHEAS), a crucial steroid hormone for adrenal function and pubertal development, is highly associated with related diseases. This study aims to develop and validate an isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (cRMP) for quantifying serum DHEAS. Serum samples were prepared by protein precipitation with acetonitrile and separated on a reversed phase column. Assay validation was conducted under the guidance of standard documents, including C62-A, EP6-A, EP10-A3, and C50-P endorsed by the Clinical and Laboratory Standards Institute (CLSI). The cRMP was established and proven to be highly specific without significant matrix effect and able to accurately quantify DHEAS in human serum. The intra-assay and inter-assay imprecision ranged from < 0.1 to 1.0% and from 1.0 to 1.1%, respectively. Trueness was assessed by recovery rate from 99.6 to 101.2%. The limit of detection (LoD) was 0.270 nmol/L, and the lower limit of the measuring interval (LLMI) was 12.1 nmol/L. A linear correlation ranged from 7.60 to 41 858 nmol/L was observed with a correlation coefficient > 0.999. R2 value of linear regression analysis between this method and clinical immunoassays was ≥ 0.990. The relative expanded uncertainty was 1.5%-2.5% over the concentration range of 12.1-33 043 nmol/L. This study developed an ID-LC-MS/MS-based cRMP, which provided high specificity, trueness, and precision for serum DHEAS quantification, contributing to the DHEAS measurement standardization and traceability.
This study investigates the presence and quantification of phthalates released into mineral waters from virgin and recycled polyethylene terephthalate (PET) bottles. A green, solvent-free analytical method based on solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC/MS) was developed and validated to detect the following four target phthalates: diethyl phthalate (DEP), diisobutyl phthalate (DiBP), dibutyl phthalate (DBP), and bis(2-ethylhexyl) phthalate (DEHP). The analytical method showed good sensitivity, precision, accuracy, and low limits of detection and quantification. Seventeen commercial water samples were analyzed, including 11 from virgin PET and six from recycled PET bottles with varying recycling content (30%-100%). Phthalate concentrations ranged from 2.21 to 18.96 μg/L, with DEHP being the most prevalent compound. Risk assessment following EFSA guidelines demonstrated that the estimated daily intakes (DIs) and the risk quotient (RQ) of all detected phthalates were well below the tolerable DI limits for both adults and toddlers. These findings confirm that the proposed method is suitable for routine screening of phthalates in mineral water and highlight the need for stricter quality controls in the recycled PET supply chain.
Phosphatidylethanols (PEth) are ethanol-derived phospholipids formed in red blood cell membranes during alcohol exposure and have emerged as highly specific biomarkers for recent alcohol use. Their extended 2-4-week detection window makes them uniquely valuable for objective monitoring in liver disease and transplantation, where accurate assessment of alcohol abstinence is critical. We developed and validated a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of PEth 16:0/18:1 and PEth 16:0/18:2 in whole blood. Multiple sample extraction strategies were evaluated to optimize recovery, sensitivity, and workflow practicality. The final method employed phospholipid-removal cartridges in a reversed-use configuration to retain, rather than remove, phospholipids. Wash and elution conditions were systematically optimized to achieve adequate sensitivity without a post-extraction evaporation step, enabling direct LC-MS/MS loading after elution and automation compatibility. Due to detectable and lot-variable endogenous PEth in commercial human blood products, multiple matrix sources were evaluated. Chicken whole blood demonstrated the cleanest background and was selected for calibrator and quality-control preparation. The developed method achieved linearity from 10 to 1000 ng/mL (R2 > 0.99) with limits of quantification below 10 ng/mL, and within- and between-run precision of < 10% and < 12%, respectively. Method comparison against a national reference laboratory showed excellent agreement (R2 ≥ 0.93; bias within ± 5%). No significant carryover, ion suppression, or lipid interference was observed. This developed and optimized LC-MS/MS method provides a sensitive, simplified, and automation-compatible approach for PEth 16:0/18:1 and 16:0/18:2 quantification, well-suited for high-throughput implementation in clinical laboratories.
Tumor progression locus 2 (TPL2, also known as MAP-3K8/COT) is a kinase that regulates MAPK and NF-κB signaling and contributes to antiviral immunity. While mammalian TPL2 has been widely studied, the TPL2-associated protein profile in swine cells remains largely unexplored. In this study, we generated an IP-LC-MS/MS-based candidate dataset of TPL2-associated proteins in porcine kidney epithelial (PK-15) cells. FLAG-tagged TPL2 was overexpressed in PK-15 cells and enriched by immunoprecipitation, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. After subtraction of proteins detected in the empty-vector negative IP control, a total of 666 candidate TPL2-associated proteins were identified. Bioinformatic analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and protein-protein interaction (PPI) network analysis, indicated that these candidates were mainly associated with RNA binding, protein modification, ubiquitin-proteasome processes, spliceosome-related pathways, and immune-associated signaling. Collectively, our results provide an IP-LC-MS/MS-based candidate resource of TPL2-associated proteins in porcine PK-15 cells, offering a useful basis for future studies on TPL2-mediated host signaling and virus-host interactions.
This tutorial provides a structured introduction to mass spectrometry (MS), with particular emphasis on electrospray ionization (ESI)-based measurements. It is intended to support researchers from chemistry and related disciplines in developing a coherent understanding of MS principles, instrumentation, and data interpretation. The architecture of mass spectrometers is outlined, including ion generation, mass analysis, and detection, together with the fundamental processes by which analytes are converted into gas-phase ions. Emphasis is placed on practical interpretation of mass spectra, including isotopic pattern analysis, molecular mass and elemental composition determination, and the evaluation of spectral peak characteristics such as resolution. The role of tandem mass spectrometry (MS/MS) in structural elucidation, particularly in the context of ESI-induced fragmentation pathways, is discussed. Terminology is aligned with IUPAC recommendations to ensure conceptual clarity. Selected illustrative examples demonstrate the integration of theoretical principles with experimental practice in compound identification and analysis.
Nonvolatile and polar fractions, largely composed of NSO heteroatom-containing compounds, play a key role in petroleum characterization by providing insights into organic geochemical parameters, such as thermal maturity and depositional environment, as well as physicochemical properties, including acidity and basicity. Electrospray ionization in the negative ion mode coupled with Fourier transform ion cyclotron resonance mass spectrometry (ESI(-) FT-ICR MS) applied to undisturbed whole oil samples has been established as a gold-standard technique for oil characterization, owing to its ability to directly ionize a broad range of polar compounds. However, the large volume and complexity of the resulting data pose significant challenges for data preprocessing, analysis, and visualization. In this study, we compared two distinct data preprocessing methodologies and show them to provide deeper insight into their application in FT-ICR MS petroleomics for inter-basin discrimination. We used lacustrine oils from two Brazilian Pre-salt basins as a case study.
Cold EI uses helium as the column carrier and cooling make-up gases at ~50 mL/min. In the rare case that helium supply could be temporarily interrupted, nitrogen or hydrogen can be used, but hydrogen leads to decomposition at the GC injector, and thus, nitrogen is preferred. However, nitrogen leads to longer analysis time and/or reduced separation. We describe the use of helium as the column carrier gas and nitrogen as the cooling make-up gas. In this "Helium Saver" mode, the helium consumption is as in standard EI and nitrogen can be used alone in stand-by mode for further helium saving.
Gas chromatography coupled with electron ionization and high-resolution mass spectrometry (GC-EI-HRMS) has been increasingly used in analytical chemistry in recent years. Databases (libraries) of such mass spectra are emerging, and various studies require processing batches of such data. This necessitates the development of user-friendly software for curation and batch processing of such data sets. In this work, we propose the "gchrmsexplain" software specifically developed for this purpose. It includes both a graphical user interface and a command-line interface. The software receives a peak list as input and finds a formula for each peak using in silico fragmentation or combinatorial enumeration. The isotopic pattern is checked. The fraction of the ion current due to the annotated peaks is calculated. The absolute error in determining m/z can be specified in either millidaltons or parts per million. Batch processing is possible. The software was applied to six publicly available GC-EI-HRMS data sets: RECETOX (exposome and metabolome libraries), HREI-MSDB, library from Castro's work (2022), and two subsets of MassBank EU. In total, these data sets contain 1811 mass spectra. Impurity peaks, systematic errors in m/z, and completely incorrect mass spectra were detected. It was shown that some of the data sets contain low-quality mass spectra. It was demonstrated that the presented software can be used for the curation of such data. The software is free and available online: https://github.com/mtshn/gchrmsexplain.
