bims-metlip 2026-09-13 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–09–13
thirty-one papers selected by
Sofia Costa, Matterworks

ExchangeXplorer: an R/Shiny application for processing and visualization of LC-HDX-MS data in metabolomics, lipidomics, and exposomics.
A Simplified and Robust LC-MS/MS Method for Simultaneous Quantification of Six Steroid Hormones Associated With Polyendocrine Metabolic Ovarian Syndrome.
MRM Processor: An Integrated Workflow for Automated MRM Data Processing via Relative Retention Time Correction.
Thiol-Ene Click-Derived Porous-Layer Open-Tubular Column for Online Purification and Multiplex LC-MS/MS Analysis of Neurotransmitters.
MetaboAnnotate: An AI-powered Multiagent Framework for Integrating Annotation Tools for Untargeted Metabolomics.
Mass spectrometry imaging in plants: Methods and applications.
LC-MS/MS Method for Simultaneous Quantification of Zolpidem, Zopiclone, and Their Metabolites in Human Urine and Its Pharmacokinetic Application.
Fast and Scalable Sample Preparation for Integrated Metabolome and Exposome-Wide Association Studies (MWAS/ExWAS).
[Rapid high performance liquid chromatography-tandem mass spectrometry method for the detection of 62 psychotropic drugs in plasma].
Improved Analysis and Characterization of Brominated Vegetable Oil in Multiple Beverages Using LC-MS/MS and LC-HRMS.
Beyond target lists: direct injection liquid chromatography-tandem mass spectrometry for comprehensive monitoring of legacy and emerging per- and polyfluoroalkyl substances in water systems.
Hierarchical mixed-effects model for batch effect correction in primary-tissue single-cell metabolomics by mass spectrometry.
Integrating dispersive liquid-liquid microextraction with HPLC-MS/MS for simultaneously determining urinary tobacco exposure biomarkers and neurodevelopmental metabolites.
Biomonitoring of parabens and PFAS in dried blood spots: An LC-QqQ-MS/MS approach including free and conjugated species.
Development and Validation of a UPLC-MS/MS Method for Simultaneous Quantification of Ensartinib and Its Major Metabolite M465 in Rat Plasma and Application to Pharmacokinetic Studies.
Non-target screening using fast comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry II: the mosaic workflow - development of a scalable and open-source non-target screening data processing workflow for LC×LC-HRMS and its application to wastewater effluent.
The data substrate of exposome intelligence: an interoperability profile for untargeted metabolomics.
A rapid isocratic LC-MS/MS method for the quantification of busulfan in human plasma and its clinical application in pediatric bone marrow transplantation.
Ultrafiltration for baclofen measurement in blood using liquid chromatography-tandem mass spectrometry.
Advances in the biomonitoring of flame retardants in human matrices.
A Fast Method to Quantify Free and Esterified Cholesterol in Human Plasma and Disease Models.
Machine Learning-Enhanced LC-HRMS Workflows for Suspect and Nontargeted Screening of Pesticide Residues in Fruits and Vegetables.
An integrated LC-MS/MS workflow for multi-matrix determination of glyphosate and aminomethylphosphonic acid for occupational exposure assessment.
Chemical standardization of kava (Piper methysticum) extract using UHPLC-MS/MS and the method of standard addition.
Dominant doubly charged ion enabling robust LC-MS/MS quantification of a novel small-molecule antibacterial drug Cefidelbaz in human plasma and its application to pharmacokinetic and protein binding studies.
A Survey of Burkholderiales Secondary Metabolites Using Library-Scale Stable Isotope Labeling Reveals Selective Production of Methyl-Esterified 3-Hydroxybutyrate Oligomers.
A novel multi-aptamer electrospun nanofibrous membrane for simultaneous selective extraction of monoamine neurotransmitters and determination in biological samples by LC-MS.
Forensic LC-MS/MS Monitoring of Anticoagulant Rodenticide Poisoning in Cats Over a Half-Decade.
Non-target screening using fast comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry I: investigating stability across a set of wastewater samples.
Expanded evaluation of a robust on-line sample cleanup-LC-MS platform suited for new approach methodologies (NAM).
"Precise-Practical-Rapid" strategy for discriminating closely related cultivars: insights from chrysanthemum varieties.

Anal Chim Acta. 2026 11 01. pii: S0003-2670(26)01016-0. [Epub ahead of print]1421 346066

ExchangeXplorer: an R/Shiny application for processing and visualization of LC-HDX-MS data in metabolomics, lipidomics, and exposomics.

Tomas Cajka, Jiri Hricko, Lucie Rudl Kulhava, Veronika Hola, Michaela Paucova, Michaela Novakova, Oliver Fiehn.

   BACKGROUND: Reliable annotation remains a major challenge in untargeted LC-MS-based metabolomics, lipidomics, and exposomics. Liquid chromatography-hydrogen/deuterium exchange-mass spectrometry (LC-HDX-MS) provides orthogonal structural information by revealing the number of exchangeable hydrogens within a molecule, thereby supporting functional-group assignment, distinguishing isomeric structures, and reducing false-positive annotations. However, broader adoption of LC-HDX-MS for small-molecule analysis has been limited by the lack of dedicated software for systematic data processing and interpretation.
RESULTS: We developed ExchangeXplorer, an open-source R/Shiny application for processing and visualizing LC-HDX-MS data from small molecules. The software accepts feature tables generated by MS-DIAL, mzmine, and related workflows, automatically pairs unlabeled and HDX-labeled features, calculates deuterium-induced mass shifts, and exports results for downstream annotation. Additional modules provide visualization of paired MS1 and MS/MS spectra, chromatographic validation using extracted ion chromatograms, estimation of exchangeable hydrogens from molecular structures, and generation of m/z-retention time target lists. Evaluation using 163 reference compounds representing metabolites, lipids, pharmaceuticals, and exposome-related chemicals showed that incorporation of experimentally determined exchangeable-hydrogen counts reduced PubChem isomer candidates by an average of 65%. Application to human plasma and serum datasets further demonstrated utility in complex biological matrices.
SIGNIFICANCE: ExchangeXplorer provides a dedicated framework for integrating HDX-derived information into untargeted LC-MS annotation workflows, improving confidence in small-molecule characterization and reducing candidate-space complexity.

Keywords:  Exposomics; Hydrogen/deuterium exchange; LC–MS; Lipidomics; Metabolomics; Structural characterization

DOI:  https://doi.org/10.1016/j.aca.2026.346066
Biomed Chromatogr. 2026 Oct;40(10): e70609

A Simplified and Robust LC-MS/MS Method for Simultaneous Quantification of Six Steroid Hormones Associated With Polyendocrine Metabolic Ovarian Syndrome.

Mengxue Feng, Yan Song, Xueqing Qian.

  Polyendocrine metabolic ovarian syndrome (PMOS), formerly known as polycystic ovary syndrome (PCOS), is characterized by dysregulation of multiple steroid hormones. Accurate quantification of these analytes is essential for laboratory evaluation; however, currently may involve complex sample-preparation workflows or provide insufficient analytical coverage, particularly for dehydroepiandrosterone sulfate (DHEAS). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was established for the simultaneous measurement of six steroid hormones associated with PMOS. Sample preparation combined liquid-liquid extraction with protein precipitation. Chromatographic separation was completed within 6 min. Validation followed international recommendations and included linearity, sensitivity, precision, accuracy, matrix effects, selectivity, and specificity. All analytes showed excellent linearity (R = 0.9959-0.9995), with intra- and inter-day CVs ≤ 5.6%. Spike recovery ranged from 85.2% to 114.9%. Accuracy was further supported by third-party QC materials and successful participation in an external quality assessment program. The assay provided an expanded linear range for DHEAS (20-20,000 ng/mL). This validated LC-MS/MS assay provides rapid, sensitive, and reproducible quantification of six PCOS-related steroid hormones and is suitable for routine laboratory implementation.

Keywords:  dehydroepiandrosterone sulfate (DHEAS); liquid chromatography–tandem mass spectrometry (LC–MS/MS); polycystic ovary syndrome (PCOS); polyendocrine metabolic ovarian syndrome (PMOS); serum; steroids

DOI:  https://doi.org/10.1002/bmc.70609
Anal Chem. 2026 09 08. 98(35): 25770-25781

MRM Processor: An Integrated Workflow for Automated MRM Data Processing via Relative Retention Time Correction.

Yao-Yu Chen, Na An, Xin-Yi Wei, Quan-Fei Zhu, Yu-Qi Feng.

Targeted metabolomics using multiple reaction monitoring (MRM) provides sensitive and selective quantification, but large-scale data processing remains challenged by retention time (RT) drift, incorrect peak detection, and subjective integration. Here, we present the MRM Processor, an integrated, automated workflow that leverages relative retention time (RRT)-driven RT correction, derivative-based signal characterization, chromatographic peak integration, and calibration-based quantification. By dynamically updating analyte RTs using designated internal standards and evaluating chromatographic signals with derivative patterns, the workflow improves peak localization while reducing the level of manual intervention. Comprehensive validation using bile acid standards, matrix-spiked biological samples, and a pediatric sepsis fecal cohort demonstrated robust RT correction, accurate peak detection, reliable quantitative performance, and superior peak classification compared with existing data processing tools. Additional validation using structurally diverse metabolite standards further defined the current applicability boundary of the RRT correction strategy. The MRM Processor is freely available and compatible with standard MRM data acquired by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

DOI: https://doi.org/10.1021/acs.analchem.6c02846
Anal Chem. 2026 09 08. 98(35): 26089-26103

Thiol-Ene Click-Derived Porous-Layer Open-Tubular Column for Online Purification and Multiplex LC-MS/MS Analysis of Neurotransmitters.

Enrico Taglioni, Andrea Cerrato, Aldo Laganà, Carmela Maria Montone, Anna Laura Capriotti.

A thiol-ene-based porous stationary phase (PLOT-type coating) was developed for online sample cleanup and enrichment coupled to LC-MS/MS for the simultaneous determination of structurally diverse neurotransmitters in biological matrices. The resulting porous thiol-ene functionalized capillary column combines the inherently high permeability of the open-tubular architecture with tailored surface functionality, enabling direct injection and efficient handling of highly polar, low-abundance analytes without derivatization. The method allows multiplexed quantification of serotonin, dopamine, glutamic acid, γ-aminobutyric acid, and reduced glutathione in plasma, cell samples, and brain tissue within a simplified analytical workflow. Compared to conventional off-line protocols, the proposed approach minimizes sample preparation while maintaining high sensitivity and reproducibility, with recoveries ranging from 86% to 100% and matrix effects within 90-104%. The platform demonstrates reliable analytical performance across different biological matrices and is compatible with routine LC-MS/MS instrumentation. Overall, this work establishes the thiol-ene-porous capillary as a viable strategy for integrated sample processing in bioanalysis and expands its application to polar metabolite profiling in complex systems.

DOI: https://doi.org/10.1021/acs.analchem.6c04601
Anal Chem. 2026 09 08. 98(35): 25617-25625

MetaboAnnotate: An AI-powered Multiagent Framework for Integrating Annotation Tools for Untargeted Metabolomics.

Yan Zhou Chen, Brandon Mukadziwashe, Frederick Zhang, Soha Hassoun.

Metabolite annotation remains a major bottleneck in untargeted metabolomics, limiting biological interpretation of large-scale mass spectrometry data sets. Although substantial advances have been made through spectral libraries, machine learning-based annotation models, and community benchmarking efforts, many recently developed tools remain difficult to incorporate into routine workflows because they are distributed as research-oriented software with complex dependencies and nonstandard interfaces. Here, we present MetaboAnnotate, a web-based framework that uses a large language model (LLM) in a multiagent system to orchestrate multiple metabolite annotation tools through a unified natural-language interface. The system enables users to submit MS/MS spectra and execute multitool annotation workflows without local installation or programming expertise. The current implementation integrates complementary methods, including SIRIUS, FLARE, JESTR, and DiffMS. Evaluation on the CASMI 2016 and CASMI 2022 benchmarks shows that agreement among independent annotation tools substantially reduces false discovery rates and improves annotation accuracy. Application to a fecal metabolomics data set further demonstrates the utility of multitool consensus for identifying high-confidence putative metabolites. MetaboAnnotate is available at: https://hassounlab.cs.tufts.edu/MetaboAnnotate/.

DOI: https://doi.org/10.1021/acs.analchem.6c01717
Curr Opin Plant Biol. 2026 Sep 09. pii: S1369-5266(26)00105-6. [Epub ahead of print]94 102962

Mass spectrometry imaging in plants: Methods and applications.

Yonghui Dong, Youwei Lu, Asaph Aharoni.

Mass spectrometry imaging (MSI) has emerged as a powerful platform for spatial metabolomics, enabling direct mapping of metabolites within plant tissues. However, successful application of MSI in plants remains strongly dependent on effective sample preparation, as the unique structural features of plant tissues pose significant analytical challenges. In this review, we highlight recent advances in sample preparation strategies designed to address these limitations across diverse plant tissues. We then discuss how the methodological and technological innovations improve spatial resolution, sensitivity, specificity, and analytical throughput. Building on this technical progress, we survey the expanding biological applications of MSI in plant science, from decoding plant-environment interactions and resolving gene function to tracing the dynamics of metabolism through stable isotope labeling. By linking chemical distributions with biological processes, MSI is playing an increasingly important role in uncovering how metabolic pathways are organized and regulated within plant tissues, ultimately enabling a spatially resolved understanding of plant metabolism.

DOI: https://doi.org/10.1016/j.pbi.2026.102962
Biomed Chromatogr. 2026 Oct;40(10): e70612

LC-MS/MS Method for Simultaneous Quantification of Zolpidem, Zopiclone, and Their Metabolites in Human Urine and Its Pharmacokinetic Application.

Varsha Chauhan, Manu Sharma, Murtada Taha, Manish Kumar, Shruti Srivastava, Naiyer Shahzad, Md Ali Mujtaba, Ameeduzzafar Zafar.

  Zolpidem (Z1) and zopiclone (Z2) are widely prescribed for insomnia; but their misuse in drug-facilitated crimes presents significant forensic and clinical challenges. This study aimed to develop and validate a simple and sensitive LC-MS/MS method, coupled with dispersive liquid-liquid microextraction (DLLME), for the simultaneous determination of Z1, Z2, and 18 phase I and phase II metabolites (20 species) in human urine. DLLME employed 1 mL of dichloromethane as extractant and 2 mL of acetonitrile as disperser per 2 mL of urine, reducing halogenated-solvent use compared with conventional liquid-liquid extraction. Chromatographic separation was achieved on a HyPURITY C18 column with water-acetonitrile (70:30, v/v) containing 0.2% formic acid. Z1 and Z2 were validated according to ICH guidelines over 0.1-200 ng/mL, showing linearity (R2 > 0.999), accuracy (96.29%-99.56%), precision (RSD ≤ 3.12%), and recovery (96.12%-98.74%). Because authentic metabolite standards were unavailable, metabolite concentrations were estimated using parent-drug calibration curves and are considered semi-quantitative. In five healthy volunteers, hydroxylated and carboxylated metabolites peaked at 2-12 h and remained detectable up to 84 h. These findings support the method's potential for retrospective urinary screening of Z-drug exposure in forensic and clinical toxicology.

Keywords:  DLLME; LC–MS/MS; drug‐facilitated crime; metabolites; method validation; urinary excretion; urine; zolpidem; zopiclone

DOI:  https://doi.org/10.1002/bmc.70612
Anal Chem. 2026 09 08. 98(35): 26044-26053

Fast and Scalable Sample Preparation for Integrated Metabolome and Exposome-Wide Association Studies (MWAS/ExWAS).

Julia Füreder, Amira Müller, Giulia Guerra, Sabina Sieri, Max Lennart Feuerstein, Benedikt Warth.

Broad coverage exposome- and metabolome-wide association studies rely on advanced instrumentation, typically anchored in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) to investigate exposure-effect associations. Sample preparation for biological fluids is a delicate matter, as it is essential to strike a pragmatic balance between sensitivity, chemical coverage, robustness, and time efficiency to meet the requirements of large-scale epidemiological studies. Here, we established a protein precipitation workflow in a scalable, high-throughput format for human plasma and urine. Different protocols were evaluated utilizing a xenobiotic mixture containing >200 exposure compounds based on extraction recovery, repeatability, and applicability for nontargeted analysis (NTA) and suspect screening. The tested high-throughput options included a protein precipitation workflow in 96-well plates and a phospholipid removal plate. For urine, a dilute-and-shoot approach was additionally tested. The plate-based protein precipitation resulted in superior performance with acceptable extraction recoveries (RE, 60-140%) for >70% of the highly diverse analyte panel in plasma and >80% in urine, and acceptable repeatability with relative standard deviations of <30% for more than 95% of analytes in plasma and 80% in urine. NTA results exhibited only a small number of altered annotated features compared to a tube-based protocol used for benchmarking, while increasing the overall time efficiency by a factor of five. To further test the protocol, it was applied to the NIST plasma standard reference material SRM1950. A total of 22 toxicants were identified and (semi-)quantified, with most concentrations comparable to available reference values. The results demonstrate the suitability of the established protocol for combined MWAS/ExWAS.

DOI: https://doi.org/10.1021/acs.analchem.6c03820
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2026 Aug 20. 44(8): 694-698

[Rapid high performance liquid chromatography-tandem mass spectrometry method for the detection of 62 psychotropic drugs in plasma].

J Ma, B Zhou, F Dong, X R Guo, C W Bi, M L Zhang, H L Li.

  Objective: To establish a rapid method for 62 psychotropic drugs in plasma using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) . Methods: In January 2025, plasma samples were extracted by methanol∶acetonitrile (1∶1, V/V), and then gradient eluted using a C18 column with an aqueous solution containing 5 mmol/L ammonium acetate and 5 mmol/L ammonium acetate in methanol as the mobile phase. Positive and negative ion rapid switching scanning modes were used, and segmented multi-reaction monitoring mode (MRM) was used for analysis. Quantification was performed by the external standard method. Results: There were a good linear relationships for 62 psychotropic drugs in plasma with correlation coefficients ranging from 0.9976 to 0.9999. The recovery rates of spiked samples at low, medium and high concentrations ranged from 90.7% to 107.9%, with relative standard deviations for intra-batch and inter-batch precision ranging from 0.9% to 11.1%. Conclusion: This method is simple, fast, and suitable for clinical poisoning detection, and for the simultaneous determination of 62 psychotropic drugs in plasma.

Keywords:  Chromatography, high pressure liquid; Plasma; Poisoning; Psychotropic drugs; Tandem mass spectrometry; Therapeutic drug monitoring

DOI:  https://doi.org/10.3760/cma.j.cn121094-20250423-00159
J Agric Food Chem. 2026 Sep 09. 74(35): 27978-27985

Improved Analysis and Characterization of Brominated Vegetable Oil in Multiple Beverages Using LC-MS/MS and LC-HRMS.

Jessica P Rafson, Christopher R Beekman, Katherine Carlos.

  Brominated vegetable oil (BVO) was previously permitted in beverages at up to 15 ppm to stabilize fruit flavorings but is no longer authorized for use in foods in the U.S. following FDA's revocation of its authorization (21 CFR § 180.30), as there is no longer a basis to conclude that this use is safe. Existing measurement methods rely on either indirect analysis requiring extensive sample preparation or direct methods using uncommon solvents or specialized equipment. This work presents a direct liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using readily available instrumentation, standard solvents, and simple sample preparation. The method monitors 9 unique BVO species, which were characterized using high-resolution MS. The LC-MS/MS BVO method has low detection limits (∼10 parts per billion), high linearity (R2 ≥ 0.995), and high accuracy (recoveries of 84-106%). Additionally, the method was tested on market soda samples listing BVO on the label.

Keywords:  LC-MS/MS; beverages; brominated vegetable oil; characterization; soda

DOI:  https://doi.org/10.1021/acs.jafc.6c07349
J Chromatogr A. 2026 Aug 26. pii: S0021-9673(26)00719-3. [Epub ahead of print]1787 467392

Beyond target lists: direct injection liquid chromatography-tandem mass spectrometry for comprehensive monitoring of legacy and emerging per- and polyfluoroalkyl substances in water systems.

Magdalena Herok, Hanna Barchańska, Monika Partyka, Maciej Chrubasik.

  Per- and polyfluoroalkyl substances (PFAS) are a large and structurally diverse group of persistent contaminants widely detected in aquatic environments, creating significant analytical and regulatory challenges. This study presents a direct-injection liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 51 PFAS, including legacy compounds and emerging alternatives, in groundwater, surface water, and tap water. By eliminating solid-phase extraction (SPE), the approach simplifies sample preparation, reduces solvent consumption, and minimises contamination risks associated with conventional workflows. The method showed consistent analytical performance, with limits of quantification ranging from 0.65 to 10 ng/L and matrix effects were maintained within ±20%, effectively corrected using isotope-labelled internal standards. Procedural blanks showed negligible contributions to target analyte signals, confirming low background contamination, while a delay column improved data reliability by separating system-derived PFAS from analyte signals. The method was suitable for a broad range of PFAS, including short-chain and structurally diverse compounds that are often difficult to quantify using traditional SPE-based methods. Overall, the proposed direct-injection LC-MS/MS method provides a reliable, efficient, and high-throughput alternative for comprehensive PFAS monitoring in tap, surface, and groundwater. By expanding analytical coverage beyond conventional targeted methods, it enables more representative assessment of PFAS occurrence and environmental risk, supporting future monitoring programmes and regulatory strategies.

Keywords:  Direct injection; Environmental monitoring; LC–MS/MS; Matrix effects; PFAS; Water analysis

DOI:  https://doi.org/10.1016/j.chroma.2026.467392
Anal Chim Acta. 2026 11 01. pii: S0003-2670(26)00992-X. [Epub ahead of print]1421 346042

Hierarchical mixed-effects model for batch effect correction in primary-tissue single-cell metabolomics by mass spectrometry.

Chunfei Zhong, Jiewei Deng, Guihua Deng, Yuehua Wu, Yunyun Yang, Chunyan Hao, Tiangang Luan.

  Single-cell mass spectrometry (MS) metabolomics is a powerful tool for profiling metabolites in primary tissues. However, daily instrumental drift and sample preparation introduce severe batch effects, which are highly intertwined with interindividual biological variation. Additionally, single cells are fully consumed after MS detection, making conventional quality control (QC) unavailable for error correction. Herein, we established a hierarchical mixed-effects correction strategy tailored to the three-level nested structure of analytical days, individual samples and single cells. A total of 1355 single cells were used for model training and 871 independent cells for validation using zebrafish liver samples. Variance decomposition revealed day-level batch effects accounted for 32.0% of total variation and sample variation for 23.3%. After correction, the average batch silhouette coefficient dropped from 0.333 to 0.081, while 98.4% of metabolite pairwise correlations were retained. Compared with ComBat, Harmony and z-score scaling, our method achieves a better trade-off between removing technical artifacts and preserving metabolic profiles. As a QC-free workflow, this approach is reliable and widely applicable to single-cell MS datasets from diverse tissues and analytical platforms.

Keywords:  Batch-effect correction; Hierarchical mixed-effects model; Mass spectrometry; QC-free batch normalization; Single-cell metabolomics

DOI:  https://doi.org/10.1016/j.aca.2026.346042
J Chromatogr A. 2026 Aug 06. pii: S0021-9673(26)00669-2. [Epub ahead of print]1787 467341

Integrating dispersive liquid-liquid microextraction with HPLC-MS/MS for simultaneously determining urinary tobacco exposure biomarkers and neurodevelopmental metabolites.

Camilla Guerrini, Maria Vinaixa, Noelia Ramírez.

  Tobacco smoke exposure (TSE) during early life is a major public health concern, as the thousands of toxic and neurotoxic compounds present in cigarette smoke can critically disrupt brain development. Dysregulation of neurotransmitter levels has been linked to oxidative damage and an increased risk of neurodevelopmental and neurodegenerative disorders. Despite its clinical relevance, simultaneous quantification of neurotransmitters and tobacco metabolites in urine remains challenging due to their broad polarity range and the complexity of the biological matrix. To address this gap, we developed a novel hydrophilic interaction ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous quantification of 21 urinary biomarkers, including three tobacco-specific biomarkers, sixteen neurotransmitters and related metabolites, and two oxidative stress biomarkers. Sample preparation combines a clean-up step prior to the metabolite extraction by dispersive liquid-liquid microextraction from only 1 mL of urine, effectively reducing matrix effects to below 10% without enzymatic hydrolysis or derivatisation. The method achieved limits of detection as low as 0.004 ng/mL for NNAL, with extraction recoveries of 83-92% for tobacco biomarkers, 69-111% for neurotransmitters, and 83-94% for oxidative stress biomarkers. Repeatability and reproducibility were below 5% for all compounds, confirming the robustness of the method. The method was successfully applied to urine samples from children living with smoking adults, revealing possible neurotransmitters and oxidative stress alterations linked to the exposure. These findings demonstrate the suitability of the proposed approach as a reliable, sensitive, and high-throughput analytical tool for large-scale biomonitoring studies investigating the early biochemical effects of tobacco smoke exposure on neurodevelopmental health outcomes.

Keywords:  Children’s urine; DLLME; HILIC-MS/MS; Neurotransmitters; Oxidative stress biomarkers; Tobacco smoke exposure biomarkers

DOI:  https://doi.org/10.1016/j.chroma.2026.467341
Anal Chim Acta. 2026 11 01. pii: S0003-2670(26)01008-1. [Epub ahead of print]1421 346058

Biomonitoring of parabens and PFAS in dried blood spots: An LC-QqQ-MS/MS approach including free and conjugated species.

Rosalía López-Ruiz, Jesús Marín-Sáez, Roberto Romero-González, Ana Rumi Fernandez, Manuel Ángel Rodríguez Maresca, Antonia Garrido-Frenich.

   BACKGROUND: Human biomonitoring requires sensitive, reliable, and minimally invasive analytical strategies for assessing exposure to endocrine-disrupting chemicals (EDCs). Dried blood spots (DBS) offer important advantages for large-scale studies due to their low sample volume requirements, simplified collection, and easier storage and transport. In this work, a high-throughput and green analytical method was developed for the simultaneous determination of parabens, including free and conjugated species, and per- and polyfluoroalkyl substances (PFAS) in human blood collected by DBS.
RESULTS: The proposed methodology combines DBS microsampling with a miniaturized 96-well plate solid phase extraction (SPE) procedure, followed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-QqQ-MS/MS). The method was successfully validated, showing good linearity (R2 > 0.982), high sensitivity, with limits of quantification ranging from 0.01 to 0.5 μg/L, satisfactory recoveries (61-120% at 0.05, 0.5 and 5 μg/L), and precision values with RSD ≤ 20%. The method was applied to 51 human DBS samples, revealing a 100% detection frequency for methylparaben (MtP, 5.3-15.1 μg/L), ethylparaben (EtP, 0.4-5.9 μg/L), propylparaben (PrP, 0.3-4.5 μg/L), perfluorobutanoic acid (PFBA, 0.9-20.2 μg/L), and perfluorooctanoic acid (PFOA, 0.4-3.8 μg/L).
SIGNIFICANCE: The developed approach provides a robust, sensitive, eco-friendly, and reliable tool for the biomonitoring of parabens and PFAS in human blood. Its low sample volume, reduced solvent consumption, and 96-well plate format support its potential application in human biomonitoring and exposome studies.

Keywords:  96-Well plates SPE; AGREEprep evaluation; Biological fluids; EDCs; PFAS; Parabens

DOI:  https://doi.org/10.1016/j.aca.2026.346058
Drug Des Devel Ther. 2026 ;20 617920

Development and Validation of a UPLC-MS/MS Method for Simultaneous Quantification of Ensartinib and Its Major Metabolite M465 in Rat Plasma and Application to Pharmacokinetic Studies.

Shiqi Jiang, Yan Chen, Lu Cao, Haoxin Fu, Peiqi Wang, Chongmeng Yang.

   Introduction: Ensartinib is a second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small cell lung cancer (NSCLC). However, its in vivo disposition and metabolic characteristics, particularly those of its major metabolite M465, remain incompletely understood. This study aimed to develop and validate a sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of ensartinib and M465 in rat plasma and to apply the method to a pharmacokinetic study in rats.
Methods: Crizotinib was used as the internal standard (IS). Plasma samples were processed by one-step protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters Acquity BEH C18 column using gradient elution with 0.1% formic acid in water and acetonitrile. The method was validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), precision, accuracy, recovery, matrix effect, stability, carry-over and dilution integrity. The validated method was subsequently applied to determine plasma concentrations of ensartinib and M465 after oral administration of ensartinib to rats.
Results: The method showed good linearity over the concentration ranges of 0.1-100 ng/mL for ensartinib and 0.05-10 ng/mL for M465, with LLOQs of 0.1 ng/mL and 0.05 ng/mL, respectively. The intra- and inter-day precision values ranged from 2.2% to 16.4%, and the accuracy ranged from -12.3% to 8.0%, meeting the acceptance criteria for bioanalytical method validation. Recovery, matrix effect, stability, carry-over and dilution integrity were all within acceptable limits. After oral administration of ensartinib to rats, the maximum plasma concentrations (Cmax) of ensartinib and M465 were approximately 64.7 ng/mL and 6.3 ng/mL, respectively. The AUC(0-t) values of ensartinib and M465 were 577.6 ng/mL*h and 32.5 ng/mL*h, respectively, indicating lower systemic exposure of M465 compared with the parent drug.
Conclusion: A sensitive, reliable, and rapid UPLC-MS/MS method was successfully developed and validated for the simultaneous determination of ensartinib and M465 in rat plasma. The method was successfully applied to a rat pharmacokinetic study and provides useful analytical support for further preclinical investigations of ensartinib and its major metabolite.

Keywords:  M465; UPLC–MS/MS; ensartinib; pharmacokinetics; rat

DOI:  https://doi.org/10.2147/DDDT.S617920
J Chromatogr A. 2026 Sep 02. pii: S0021-9673(26)00748-X. [Epub ahead of print]1787 467422

Non-target screening using fast comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry II: the mosaic workflow - development of a scalable and open-source non-target screening data processing workflow for LC×LC-HRMS and its application to wastewater effluent.

Oskar Munk Kronik, Ryland T Giebelhaus, Jan H Christensen, Selina Tisler, Giorgio Tomasi, Nikoline Juul Nielsen.

  Comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry (LC × LC-HRMS) offers improved group-type separation and mass spectral quality than one-dimensional LC-HRMS, but its widespread adaption remains limited, partly, due to labor-intensive and non-scalable data-processing workflows. Here, we present Mosaic, an open-source NTS workflow for LC × LC-HRMS that groups ions originating from a single compound, including adducts, isotopes, and in-source fragments, into single components within and across samples. Mosaic was evaluated using three componentization strategies: mass filtering (MF), non-negative matrix factorization (NMF), and their combination (MF+NMF). The workflow was validated using 78 LC × LC-HRMS chromatograms originating from wastewater effluents samples from six wastewater treatment plants, quality control samples, field blanks and three instrument blanks. Fourteen internal standards (IS), present in 75 chromatograms, were used to quantify detection performance, across-sample grouping efficiency, and the accurracy of automated peak volume extraction. Within-sample true positive rates of detection (TPRs) were 52% (NMF), 91% (MF), and 99% (MF+NMF), respectively. All detected IS were componentized correctly for the MF+NMF. Only the combined MF+NMF componentization approach could extract peak volumes comparable to those obtained by manual integration (p = 0.99). This performance relied on the generation of higher purity mass spectral loadings, achieved through correlation-based removal of interfering ions in both chromatographic dimensions as done in MF, and NMF's ability to resolve peaks and corresponding ions with varying relative abundances across samples. The Mosaic workflow provides an open-source, transparent, and scalable NTS solution for complex samples analysed by LC × LC-HRMS, enabling reliable detection and peak volume extraction for trace-level contaminants at ng/mL concentrations.

Keywords:  CHEMOMETRICS; LC×LC-HRMS; Mass filtering; Non-target screening; Wastewater effluent

DOI:  https://doi.org/10.1016/j.chroma.2026.467422
Front Artif Intell. 2026 ;9 1901969

The data substrate of exposome intelligence: an interoperability profile for untargeted metabolomics.

Thomas Hartung.

  Untargeted metabolomics, anchored in high-resolution mass spectrometry, has matured into the central analytical platform of human exposomics. It can capture endogenous biology, diet, drugs, microbial chemistry, environmental contaminants, and their transformation products from a single biological sample. Yet exposome science remains stubbornly single-study: most untargeted exposomics publications stand alone, featuring tables, partial annotations, and semi-quantitative intensities that cannot be combined across cohorts. The bottleneck is no longer instrumentation or annotation; it is interoperability. Existing standards, including MSI, mQACC, BP4NTA, NORMAN, MERIT, mzML, mzTab-M, ISA-Tab, the Universal Spectrum Identifier, RefMet, ChEBI, MetaboLights, Metabolomics Workbench, GNPS/MassIVE, and the emerging GA4GH human exposome data standards, cover the necessary ingredients but do not yet compose a single, executable profile. I argue that the next stage of exposomics must move from FAIR deposition to meta-analysis-ready evidence: a four-layer stack of acquisition comparability, machine-readable reporting, evidence-aware annotation, and standardized summary statistics, validated by a living community benchmark. Cumulative exposome science depends on it.

Keywords:  FAIR data; data interoperability; exposome; federated learning; high-resolution mass spectrometry; meta-analysis; metabolite annotation; untargeted metabolomics

DOI:  https://doi.org/10.3389/frai.2026.1901969
Anal Methods. 2026 Sep 11.

A rapid isocratic LC-MS/MS method for the quantification of busulfan in human plasma and its clinical application in pediatric bone marrow transplantation.

Ying Xia, Yong Zhang, Feng Chen.

A rapid isocratic LC-MS/MS method for quantifying busulfan in human plasma was developed and validated. Plasma samples were processed by protein precipitation with acetonitrile using busulfan-d8 as the internal standard. Separation was performed on a Kinetex C18 column (2.1 × 50 mm, 5 µm; Phenomenex) with isocratic elution (water : acetonitrile = 70 : 30, v/v, containing 1 mM formic acid and 1 mM ammonium acetate) at 0.5 mL min-1, with a total run time of 1 min. Detection was conducted using positive electrospray ionization in multiple reaction monitoring mode (m/z 264.1 → 151.1 for busulfan; m/z 272.1 → 159.1 for busulfan-d8). The assay was linear over the concentration range of 0.100-10.0 µg mL-1 (r = 0.9998). Intra- and inter-batch RSD values were <5.5% and bias ranged from -3.6% to 5.0%. Recovery and matrix effect values were 100.1-101.3% and 101.4-101.8%, respectively. The method was successfully applied to 85 pediatric patients undergoing bone marrow transplantation. AUC0-∞ could be reliably estimated in 70 of the 85 patients (82.4%). Of these, 61.4% achieved the target AUC0-∞ (900-1500 µmol min L-1) after the first dose, while 18.6% fell below and 20.0% exceeded the target range, highlighting the substantial pharmacokinetic variability and the inadequacy of weight-based dosing in pediatric patients. This simple, rapid (1 min per sample) method is suitable for busulfan therapeutic drug monitoring in pediatric patients.

DOI: https://doi.org/10.1039/d6ay01474g
Leg Med (Tokyo). 2026 Sep 07. pii: S1344-6223(26)00223-3. [Epub ahead of print]86 102995

Ultrafiltration for baclofen measurement in blood using liquid chromatography-tandem mass spectrometry.

Akiko Takeuchi, Kotaro Matoba, Ryo Uozumi, Manabu Murakami, Takayuki Kiuchi, Shigeki Jin.

  The accurate measurement of baclofen concentrations in blood is essential for diagnosing baclofen-related fatalities, which have increased in recent years. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of baclofen in whole blood and serum using liquid chromatography-tandem mass spectrometry following ultrafiltration. This method is based on multiple reaction monitoring in positive-ion mode and has high sensitivity with a lower limit of quantification of 0.2 ng/mL. The precision and accuracy of this method satisfy the basic requirements for quantitative analysis (intra- and inter-day assays yielded relative standard deviation of ≤9.0%; the matrix effect ranged from 99.7%-105.4%). Using our method, baclofen concentrations of 442-493 ng/mL in blood samples and 62 mg/mL in urine were detected in cases of prolonged baclofen poisoning. No differences were observed between baclofen concentrations in whole blood and serum.

Keywords:  Baclofen; Forensic toxicology; Liquid chromatography–tandem mass spectrometry; Ultrafiltration

DOI:  https://doi.org/10.1016/j.legalmed.2026.102995
J Chromatogr A. 2026 Aug 29. pii: S0021-9673(26)00723-5. [Epub ahead of print]1787 467397

Advances in the biomonitoring of flame retardants in human matrices.

Zhenzhu Xue, Rui Zhou, Hong Wu, Rui Feng, Yuqing Xia, Yutong Guo, Kaiyong Liu, Sheng Wang.

  Flame retardants (FRs) represent a class of typical persistent organic pollutants extensively employed in industrial applications, yet they confer significant biological toxicity and pose substantial health risks. The quantitative determination of FRs and their metabolites in human specimens furnishes critical data for evaluating exposure levels and concomitant health risks. The selection of appropriate sample pretreatment and detection methods depends on the specific FR class and the physicochemical characteristics of the biological matrix. This review provides a comprehensive synthesis of contemporary analytical strategies for FR quantification in human matrices, encompassing: (ⅰ) FR classification, (ⅱ) systematic analytical workflows, (ⅲ) matrix-specific sample preparation protocols, (ⅳ) comparative evaluation of instrumental techniques, and (ⅴ) delineation of critical methodological challenges. Emphasis is placed on method performance characteristics, matrix effects, and quality control. The objective is to provide a practical reference framework for developing robust, sensitive, and high-throughput methods for FR biomonitoring.

Keywords:  Flame retardants; Human bio-matrices; Mass spectrometry; Matrix effects; Quality control; Sample preparation

DOI:  https://doi.org/10.1016/j.chroma.2026.467397
J Lipid Res. 2026 Sep 10. pii: S0022-2275(26)00168-9. [Epub ahead of print] 101138

A Fast Method to Quantify Free and Esterified Cholesterol in Human Plasma and Disease Models.

Michaela Chocholoušková, Wai Kin Tham, Menglan He, Woon-Puay Koh, Chew-Kiat Heng, David L Silver, Federico Torta.

  Cholesterol exists in two forms in the human body: free and esterified as cholesteryl esters (CE). Free cholesterol is an essential component of cell membranes and participates in the biosynthesis of bile acids and steroid hormones. Cholesterol is transported as CE in lipoprotein particles and stored in cells as part of lipid droplets. Here, we present a rapid method (4 min) capable of analysing both cholesterol forms (free and esterified) in a single experiment, utilizing supercritical fluid chromatography (SFC) coupled with high-resolution mass spectrometry (QTOF). This method takes advantage of the commonly observed, yet often undesired, in-source fragmentation of cholesteryl esters during electrospray ionization (ESI). In addition, it offers the advantage of measuring the abundance of molecular CE species and it is suitable for low-volume samples as it can effectively analyse plasma or serum diluted up to 100-fold, requiring only a simple protein precipitation step for sample preparation. The method was validated using human plasma reference materials, showing excellent accuracy as demonstrated by the analysis of the NIST Standard Reference Material (SRM) 1950. Furthermore, the performances of this workflow for biomedical studies were tested in a large human Asian cohort for Coronary Heart Disease and in a cellular model of Niemann-Pick disease type C1 (NPC1), to follow separate pathways of cellular cholesterol metabolism.

Keywords:  Esterified and Total Cholesterol; Free; High Resolution Mass Spectrometry; Lipidomics; Supercritical Fluid Chromatography

DOI:  https://doi.org/10.1016/j.jlr.2026.101138
Anal Chem. 2026 09 08. 98(35): 25557-25574

Machine Learning-Enhanced LC-HRMS Workflows for Suspect and Nontargeted Screening of Pesticide Residues in Fruits and Vegetables.

Sergio González, Gabriel Merino, Jairo Arturo Guerrero Dallos.

Analytical methodologies are essential for detecting and quantifying contaminants. While methods for known compounds are well established, identifying unexpected or unknown compounds remains challenging due to the lack of reference data. Several strategies have been proposed to integrate analytical information into data analysis workflows, but their implementation often requires programming skills and results are rarely presented in a format familiar to analytical chemists, such as the uncertainty budgets used in quantitative analysis. We developed an integrated workflow for suspect screening and nontargeted identification of pesticides in fruits and vegetables using QuEChERS extraction and HPLC-ESI-HRMS (Orbitrap) analysis. The workflow was developed using variable data-independent acquisition (vDIA) data and evaluates protonated and deprotonated species for compound identification. The workflow estimates missing identification properties using machine learning and combines their contributions into a single confidence value. Validation with fortified matrix extracts at different mass concentrations and with real samples containing pesticide residues showed performance comparable to existing software, particularly improving nontargeted identification at high concentrations (>6% increase in identified compounds). This approach reduces the input required for suspect screening and estimates properties for candidate compounds in nontargeted analysis. Results are reported with explicit consideration of the influence of identification properties, analogous to uncertainty budgets in chemical metrology. This workflow improves the interpretability and reliability of chemical identification and supports data-driven decision-making in routine analysis.

DOI: https://doi.org/10.1021/acs.analchem.5c06726
J Chromatogr A. 2026 Sep 01. pii: S0021-9673(26)00743-0. [Epub ahead of print]1787 467417

An integrated LC-MS/MS workflow for multi-matrix determination of glyphosate and aminomethylphosphonic acid for occupational exposure assessment.

Shuping Qiang, Mahima Seeborun, Albert L Juhasz, Peter Hoffmann.

  Glyphosate, a widely used herbicide in agricultural and forestry settings, raises significant concerns regarding potential occupational exposure. However, comprehensive exposure assessment integrating biological and environmental monitoring remains limited, partly due to the lack of analytical workflows capable of supporting multiple sample matrices on a single platform. Here, we developed and validated a high-throughput derivatization-free LC-MS/MS workflow for the determination of glyphosate and aminomethylphosphonic acid (AMPA) in urine, surface wipes, patches and breath zone air monitoring samples collected using Occupational Safety and Health Administration (OSHA) versatile sampler (OVS) tubes. The workflow combines selective solid-phase extraction (SPE) using AFFINIMIP molecularly imprinted polymer cartridges for urine with a simplified universal aqueous extraction workflow for environmental matrices, enabling efficient analysis of diverse matrices using a single analytical platform. Chromatographic separation was achieved on a mixed-mode Obelisc N column within 6 minutes, with the in-sample addition of 0.1 mmol L-1 methylenediphosphonic acid (MA) significantly improved peak shape and sensitivity. The method demonstrated excellent specificity, accuracy and precision across all matrices. The limits of detection (LODs) for glyphosate and AMPA were 0.1 and 0.2 ng mL-1 in urine, respectively; and 1.6 ng in surface wipes and patches, and 0.4 ng in OVS air samples for both analytes. The method was successfully applied to 1121 biological and environmental samples collected from forestry workers in Australia, confirming its suitability for real-world exposure assessment. By integrating biomonitoring and environmental monitoring within a single analytical platform, this approach provides a practical and high-throughput tool for comprehensive assessment of occupational glyphosate exposure.

Keywords:  Aminomethylphosphonic acid; Glyphosate; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Molecularly imprinted polymer; OSHA versatile sampler (OVS); Occupational exposure; Urine

DOI:  https://doi.org/10.1016/j.chroma.2026.467417
Pharm Biol. 2026 Dec;64(1): 1034-1044

Chemical standardization of kava (Piper methysticum) extract using UHPLC-MS/MS and the method of standard addition.

Ruth N Muchiri, Natalie R Thun, Richard B van Breemen.

   BACKGROUND: Kava (Piper methysticum G.Forst.) is used traditionally in the South Pacific for ceremonial and medicinal purposes and as a dietary supplement for its anxiolytic effects. The pharmacological activity of kava is attributed to kavalactones and flavokavains, including desmethoxyyangonin, 7,8-dihydrokavain, kavain, methysticin, 7,8-dihydromethysticin, and yangonin. The safe and effective use of kava dietary supplements depends on botanical authentication and standardization to these active constituents.
OBJECTIVE: To support safety studies being carried out by the Botanical Safety Consortium, the chemical profile of a botanically authenticated kava extract was obtained using ultrahigh-pressure liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS), and kavalactones and flavokavains were identified by comparison with standards.
MATERIALS AND METHODS: The kava extract was chemically standardized using two analytical methods based on UHPLC-MS/MS with selected reaction monitoring on a triple quadrupole mass spectrometer. One method used external calibration with standards dissolved in neat solvent due to the absence of blank kava matrix, while the second used the method of standard addition to evaluate kava extract matrix effects that might affect the accuracy of the external calibration method.
RESULTS: Matrix-induced ion suppression was observed for desmethoxyyangonin (23.2%) while signal enhancement was observed for 7,8-dihydrokavain (30.1%) and flavokavain A (43.2%).
DISCUSSION AND CONCLUSIONS: The method of standard addition is ideal for correcting for botanical matrix effects that can interfere with quantitative assays based on electrospray (U)HPLC-MS/MS. Chemical authentication and standardization are essential for quality control of kava dietary supplements and to support research on their safety and efficacy.

Keywords:  Kava; Piper methysticum; electrospray UHPLC-MS/MS; matrix effects; method of standard addition; quantitative analysis

DOI:  https://doi.org/10.1080/13880209.2026.2727761
J Pharm Biomed Anal. 2026 Sep 07. pii: S0731-7085(26)00393-6. [Epub ahead of print]282 117725

Dominant doubly charged ion enabling robust LC-MS/MS quantification of a novel small-molecule antibacterial drug Cefidelbaz in human plasma and its application to pharmacokinetic and protein binding studies.

Chenxin Ding, Xiuyang Li, Yijue Wu, Jun Wang, Shuai Wang, Yiya Wang, Li Ding.

  Cefidelbaz is a novel siderophore cephalosporin antibacterial agent with a unique "Trojan horse" mechanism, which can combat some of the most difficult-to-treat Gram-negative bacterial infections. In mass spectrometric analysis, cefidelbaz generated distinct precursor ions in both positive and negative modes during the first-quadrupole full-scan (Q1 full-scan), while multiple precursor ions with different charge states were observed in positive mode. Notably, singly charged precursor ions in either positive or negative mode failed to provide wide linear range and high sensitivity, whereas the doubly charged precursor ions enabled reliable and reproducible quantitative analysis. These findings, together with reported charge-state behavior of cephalosporins with zwitterionic character, demonstrated that the optimal precursor ions should be selected according to experimental quantitative performance. Here, two independent LC-MS/MS (liquid chromatography-tandem mass spectrometry) methods were developed. The first method was established for the quantification of total cefidelbaz in human plasma, with a calibration range of 1.00-600 µg/mL. The second method, based on ultrafiltration coupled with LC-MS/MS, was developed for the determination of plasma protein binding (%PPB), covering a calibration range of 0.500-250 µg/mL. Overall, this study demonstrated the importance of precursor ions selection for reliable LC-MS/MS quantification of cephalosporins with zwitterionic character and provides validated bioanalytical methods to support clinical development of cefidelbaz.

Keywords:  Cefidelbaz; Clinical pharmacokinetics; LC-MS/MS; Plasma protein binding; Siderophore antibiotic

DOI:  https://doi.org/10.1016/j.jpba.2026.117725
J Ind Microbiol Biotechnol. 2026 Sep 10. pii: kuag026. [Epub ahead of print]

A Survey of Burkholderiales Secondary Metabolites Using Library-Scale Stable Isotope Labeling Reveals Selective Production of Methyl-Esterified 3-Hydroxybutyrate Oligomers.

Liana Zaroubi, Stephanie C Heard, Matthew Sutherland, Alessandra S Eustaquio, Roger G Linington.

  Large-scale untargeted bacterial metabolomics studies are often challenging to interpret, particularly for natural product discovery. Complex raw mass spectrometry data contains media components, biotransformation products, workup contaminants, and in-source fragments, making it difficult to resolve true biosynthesized bacterial products from background features. Incorporating stable isotope labels into untargeted workflows enables the selective detection of actively biosynthesized compounds, drastically reducing candidate mass spectrometry features to highlight novel or robustly expressed metabolites. Recently, our lab introduced IsoAnalyst, a parallel stable isotope labeling platform that links secondary metabolites to biosynthetic gene clusters by determining the incorporation rates of a panel of isotopically labeled building blocks. In this study we applied the IsoAnalyst protocol to a library of 114 Burkholderiales strains with the goals of profiling the specialized metabolite chemistry of this bacterial order and connecting detected natural products to their cognate BGCs based on their labeling profiles. While our strain-matching protocol, which evaluates correlations between detected metabolites and BiG-SCAPE-defined gene cluster families, did not yield confident BGC assignments, it successfully highlighted widely distributed metabolites with distinct labeling profiles. We targeted a set of these molecules for isolation, revealing two classes of compounds derived from primary biomolecule metabolism that are implicated in cellular stress responses. The first class of molecules were identified as lysophosphatidyl ethanolamine analogues while the second was characterized via NMR spectroscopy and mass spectrometry as methyl-esterified 3-hydroxybutyrate oligomers (HB6-OMe, HB7-OMe, and HB8-OMe), representing higher-degree polymer units than previously reported.

Keywords:  Burkholderiales; Stable isotope; gene cluster family; library-scale; metabolomics

DOI:  https://doi.org/10.1093/jimb/kuag026
Anal Bioanal Chem. 2026 Sep 10.

A novel multi-aptamer electrospun nanofibrous membrane for simultaneous selective extraction of monoamine neurotransmitters and determination in biological samples by LC-MS.

Xijia Zhang, Junfeng Qin, Chen Ma, Ningning Wang, Yufeng Li, Juanjuan Zhao, Dongsheng Li.

  Dopamine (DA) and serotonin (5-HT) are key monoamine neurotransmitters, the sensitive determination of which in biological matrices is challenging owing to low concentrations and strong matrix interference. In this study, a multi-aptamer-functionalized electrospun membrane (MA-Au@PVP/PVDF) was developed by simultaneously immobilizing the DA aptamer (Apt-DA) and the 5-HT aptamer (Apt-5-HT) on the membrane surface via Au-S bonds. The membrane combines high specific surface area, good stability, and aptamer-based specificity, enabling simultaneous and selective extraction of DA and 5-HT from complex samples. The enriched analytes were subsequently quantified using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). The maximum adsorption capacities of the MA-Au@PVP/PVDF membrane were 190.8 ng/cm2 for 5-HT and 165.1 ng/cm2 for DA, with the adsorption process fitting well to the Langmuir isotherm model (R2 ≥ 0.99). Under optimized extraction conditions, the established method was thoroughly validated in both serum and urine matrices, exhibiting wide linear ranges (2.5-1000 ng/mL, R2 ≥ 0.9905), low limits of quantification (LLOQ 2.5-10 ng/mL), satisfactory intra- and inter-day precision (RSD < 10.54%), and negligible matrix effects (86.37%-108.92%). The recoveries in serum and urine were 88.91%-103.22%, demonstrating its good broad applicability in different matrices. The proposed method was successfully applied to the determination of DA and 5-HT in human urine samples, providing a reliable and efficient pretreatment strategy for trace neurotransmitter analysis.

Keywords:  Biological sample pretreatment; Electrospun membrane; LC-Q-TOF-MS; Monoamine neurotransmitters; Multi-aptamer; Selective extraction

DOI:  https://doi.org/10.1007/s00216-026-06777-8
Vet Med Sci. 2026 Sep;12(5): e71209

Forensic LC-MS/MS Monitoring of Anticoagulant Rodenticide Poisoning in Cats Over a Half-Decade.

HyunYoung Chae, Jae-Won Byun, So-Jeong Yim, Bok-Kyung Ku, Tae-Wan Kim, JeongWoo Kang.

   BACKGROUND: Anticoagulant rodenticides (ARs) are important toxicants in veterinary forensic investigations involving suspected poisoning in cats.
OBJECTIVES: This study aimed to develop and validate a standardized LC-MS/MS-QTRAP method for the simultaneous identification and quantification of eight ARs in gastric matrices from cats and to assess its applicability to veterinary forensic toxicology.
METHODS: Automated sample preparation was implemented to reduce inter-analyst variability. Dual confirmation using multiple reaction monitoring (MRM) and enhanced product ion (EPI) spectra was applied to improve identification reliability and minimize false-positive results caused by structurally related compounds. The method was applied to 747 cat forensic toxicology cases submitted between January 2020 and August 2025, in which gastric tissue or gastric contents were available for analysis.
RESULTS: ARs were detected in 2.5% of cases (19/747). Coumatetralyl was the most frequently detected AR, followed by flocoumafen and bromadiolone. Flocoumafen was detected despite not being an officially registered active ingredient in Korea, highlighting the need for broad multi-analyte screening.
CONCLUSIONS: The standardized LC-MS/MS-QTRAP method enabled reliable detection and quantification of ARs in gastric matrices from cats. This approach provides useful forensic evidence of recent AR exposure in suspected poisoning cases and may support improved toxicovigilance for registered and unregistered rodenticides.

Keywords:  QTRAP; QuEChERS; coumatetralyl; gastric contents; toxicovigilance

DOI:  https://doi.org/10.1002/vms3.71209
J Chromatogr A. 2026 Aug 31. pii: S0021-9673(26)00734-X. [Epub ahead of print]1787 467408

Non-target screening using fast comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry I: investigating stability across a set of wastewater samples.

Oskar Munk Kronik, Ryland T Giebelhaus, Selina Tisler, Jan H Christensen, Giorgio Tomasi, Nikoline Juul Nielsen.

  Comprehensive two-dimensional liquid chromatography-high-resolution mass spectrometry (LC × LCHRMS) provides high separation capacity, but its routine use in environmental non-target screening (NTS) remains limited. A key unresolved question is whether LC × LCHRMS methods can maintain retention-time precision, peak width, and signal stability over large analytical sequences. In this study, the long-term analytical stabilility of a LC × LCHRMS method applied to effluent wastewater and point source samples collected upstream of wastewater treatmeant plant, quality control and blank samples. The method provided a corrected two-dimensional peak capacity of 1.63 × 103 (σ: 112) and a peak production rate of approximately 40 min⁻¹. Stability was assessed for a chromatographic sequence with 96 injections, corresponding to >106 h of continuous run time, using 14 isotope-labelled internal standards spiked into 75 samples. For the 12 internal standards retained in both dimensions, retention-time precision was high, with mean relative standard deviation (RSD) of 0.7% in the first dimension and 0.6% in the second dimension. Second-dimension peak broadening was minor: the median increase during the sequence was 4.4 × 10⁻² s for the 14 internal standards, corresponding to less than one HRMS scan point and approximately 6% of the median second-dimension peak width. Signal intensities for retained internal standards were also stable, with an average RSD of 6.8%. The results demonstrates that LC × LCHRMS can maintain high chromatographic efficiency and satisfactory analytical stability over extended wastewater NTS sequences. Overall, the study supports LC × LCHRMS as a platform for detailed characterization and comparison of complex environmental samples.

Keywords:  Environmental monitoring; High analytical stability; LC×LC-HRMS; Maintained high column efficiency; Retention-time stability

DOI:  https://doi.org/10.1016/j.chroma.2026.467408
Anal Bioanal Chem. 2026 Sep 08.

Expanded evaluation of a robust on-line sample cleanup-LC-MS platform suited for new approach methodologies (NAM).

Marcus Kamande, Julie Bekkhus, Stian Kogler, Aysan Safavi, Mads T S Gisle Johnsen, Patrik Appelblad, Steven Ray Wilson, Hanne Røberg-Larsen.

  Sample preparation in bioanalysis can require significant numbers of (manual) steps and consumables. Such procedures can be bottlenecks regarding cost, throughput, and greenness. This is also the case for analysis of new approach methodologies (NAMs) such as organoids and organ-on-a-chip systems. In this context, approaches tailored for measuring drugs in NAM-related cell culture media (CCM) are being developed. For mass spectrometry-based analysis, automated filtration/filter backflush solid-phase extraction liquid chromatography AFFL-SPE-LC is utilized. Case studies have shown that the "AFFL" platform allows for vast reductions in sample preparation efforts, as the "self-cleaning" filtration features and SPE conditions remove potentially clogging/contaminating materials from samples of biological origin, e.g., the salt and protein content in CCM. Here, broader demonstrations of the AFFL platform's traits for CCM analysis are provided, employing an extended panel of hydrophobic small-molecule drugs. The investigated AFFL platform shows reproducible chromatographic performance, as well as fit-for-purpose inter-matrix and inter-column robustness. Hundred-scale injections can be performed with satisfactory repeatability (e.g., retention time RSDs < 1%), with limited interference from the various CCM matrices investigated. Practical NAM applications are also demonstrated. The approach has clear advantages in greenness, obtaining an AGREE prep score of 0.71, contrasting the < 0.55 scores of more conventional/commercial approaches. The plastic consumable usage of our approach is 28 g/100 samples, a nearly 20-fold improvement over a previously established benchmark (500 g/100 samples). Taken together, these results demonstrate that AFFL provides a robust, low-waste, and scalable LC-MS-based workflow for chemical analysis of NAM-derived samples.

Keywords:  Automation; Cell culture medium; Inline; LC-MS; NAM; Solid-phase extraction

DOI:  https://doi.org/10.1007/s00216-026-06775-w
J Chromatogr A. 2026 Aug 14. pii: S0021-9673(26)00691-6. [Epub ahead of print]1787 467363

"Precise-Practical-Rapid" strategy for discriminating closely related cultivars: insights from chrysanthemum varieties.

Qian Meng, Ziqing Li, Tingwei Li, Yun Li, Hua Qu, Wenlong Wei, Qirui Bi, Jianqing Zhang, De-An Guo.

  The Chrysanthemum genus comprises six predominant varieties (Chuju, Boju, Gongju, Hangju, Huaiju, Jinsihuangju). However, their highly similar morphological and chemical characteristics make accurate discrimination challenging. To address this, a 'Precise-Practical-Rapid' integrated strategy for origin discrimination was developed, incorporating LCHRMS metabolomics (precise), polyphenol-specific HPLC fingerprinting and quantitative analysis (practical), and direct-infusion mass spectrometry and infrared spectroscopy methods (rapid). LC-QTOF MS-based metabolomics identified 221 compounds across six varieties, successfully distinguished all geographical origins, and revealed 13 key discriminatory markers. HPLC-based polyphenol-specific fingerprints and quantitative profiles achieved clear origin clustering, with a minimal four-marker panel (luteolin, rutin, 3-caffeoylquinic acid [3-CQA], and 5-CQA) retaining full discriminatory power. DI-QDa MS and FT‑IR spectroscopy enabled rapid (2 min per sample) high-throughput screening, with t‑SNE, PLS‑DA, and hierarchical cluster analysis achieving successful discrimination. Collectively, these platforms provided complementary resolution tiers: LC-QTOF MS for discovery and traceability in complex matrices, HPLC for routine quality control with reference-standard quantification, and DI-QDa or FT‑IR for on-site screening. This multi-platform comparison establishes a practical decision framework for Chrysanthemum authentication and offers a transferable pipeline for origin verification of other botanicals.

Keywords:  Authentication; Chrysanthemum; Geographical origin; Multidimensional; Profiling

DOI:  https://doi.org/10.1016/j.chroma.2026.467363