bims-metlip 2026-04-26 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–04–26
34 papers selected by
Sofia Costa, Matterworks

Practical considerations for optimizing untargeted metabolomics with Synapt XS HRMS: A tutorial.
METLIN 960 K: An Empirical Tandem Mass Spectrometry Data Resource.
Development and validation of a green DLLME-LC-MS/MS method for apixaban quantification in human plasma using Box-Behnken design optimization with application to pharmacokinetic studies and sustainability assessment.
A Simple, Rapid, and Clinically Transferable LC-MS/MS Method for the Detection of Sex Steroid Hormone Metabolic Profiles.
A rapid UPLC-MS/MS method for therapeutic drug monitoring of bupivacaine: Validation and clinical application in perioperative analgesia.
Confident Structural Annotation of Bile Acid Isomers Combining Electron-Activated Dissociation or Ion Mobility with High-Resolution Mass Spectrometry in Metabolomics.
Structure-informed deep generation enables de novo metabolite annotation in untargeted metabolomics.
Miniaturized chromatography-based lipidomics methods towards single-cell analysis.
Targeted Comprehensive Screening of Neuropsychiatric Drugs in Serum by LC-MS/MS: Method Development and Three-Year Clinical Utility.
A polarity-based two-dimensional LC-MS/MS distribution strategy combined with ion identity molecular networking for enhanced metabolite annotation in Astragalus Radix species.
An Accurate Method Based on Ultra-High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry for the Quantification of Five Cannabinoids in Juvenile and Adult Caenorhabditis elegans.
A Novel Workflow for Fast Elucidation of Drug Metabolites for Screening-Combining In Silico Metabolite Prediction With Trapped Ion Mobility QTOF-MS.
FlashMRM: An Automated Platform for MRM Method Generation and Iterative Optimization Based on High-Resolution Spectra.
A green analytical method for identification and quantification of carcinogenic impurity N-nitroso-desformyl riociguat in riociguat formulations by UHPLC-MS/MS.
Evaluation and clinical application of an accurate isotope dilution liquid chromatography-tandem mass spectrometry method for serum glycocholic acid.
Multidimensional and Hybrid Separation Strategies for Body Fluids and Tissues: Comparative Performance and Translational Considerations.
Quantification of the Active Metabolites 4-Methylaminoantipyrine and 4-Aminoantipyrine of Dipyrone From Human Plasma by LC-MS/MS.
Development and validation of an UHPLC-MS/MS method for simultaneous quantification of Tazobactam, Meropenem, Vancomycin, Linezolid and Piperacillin in human plasma: Application in critically ill patients.
Evolving LC-MS hyphenations: strengths, limitations, and emerging trends in bioanalytical applications.
CASMDB: An Open-Source Database of Metabolite Annotation Data for 1D 1H NMR-Based Metabolomics.
Development of a multi-targeted metabolomics platform for semi-quantification of faecal metabolites: a proof-of-concept analysis in human faeces.
Metabolic and lipidomic stability in EDTA plasma from hospitalized patients: impact of refrigerated storage time and additional freeze-thaw cycle.
Curation of Mass Spectrometry Reference Data for Improved Identification and Dereplication of Cyanobacterial Specialized Metabolites.
Dark Reactions in Microdroplets Explain Widespread Artifacts in Metabolomic Profiling.
Automated solid-phase extraction coupled with heart-cutting two‑dimensional liquid chromatography for simultaneous quantification of thirteen fat-soluble vitamins in foods for special medical purposes.
A Comparison of Three Analytical Procedures for Oxandrolone Long-Term Metabolite Determination in Urine Samples.
3D-printed removable-cap device with embedded magnet for magnetic solid phase extraction.
A Universal Framework for Blood Ionome Extraction and Intelligent Quality Control in 1H NMR Metabolomics.
Detection of Microbiological Contamination and Malfunctions in Technical Equipment Used in the Milk Processing Industry Using Acyl-Homoserine Lactones With Liquid Chromatography/Triple Quadrupole Mass Spectrometry.
Heart-Cutting Two-Dimensional Liquid Chromatography-Isotope Ratio Mass Spectrometry for Compound-Specific δ13C Analysis of Water-Soluble B Vitamins in Complex Supplement Matrices.
Direct Comparison of MRM-MS and PRM-MS Methods for Quantitative Ganglioside Analysis.
CorrelandSW: Correlation Networks from LC-MS Data.
Solvent-assisted sensing ability of 19F reporter in simultaneous discrimination and quantification of proline and 4-hydroxyproline isomers.
Dry Deposition Strategies for MALDI-MS Imaging: Principles, Advances and Emerging Applications.

J Chromatogr A. 2026 Apr 14. pii: S0021-9673(26)00321-3. [Epub ahead of print]1778 466991

Practical considerations for optimizing untargeted metabolomics with Synapt XS HRMS: A tutorial.

Lars Kaiser, Oliver Riester, Hans-Peter Deigner.

  Metabolomics, the systematic study of small molecules in biological samples, has rapidly emerged as a key discipline for understanding metabolism across organisms. LCHRMS displays the dominant technique for global metabolomics due to its high resolution and fast detection. In addition, some instruments offer the option of separating analytes based on their mobility in a carrier gas (ion mobility separation). However, transferring existing protocols between instruments is often limited by differences in hardware, ion mobility capabilities, and instrument generations. In particular, traveling wave ion mobility spectrometry (TWIMS) can induce ion heating and fragmentation, primarily affecting labile polar metabolites (<500 Da), requiring individual adjustment of instrument settings. Because of component redesigns, parameters of previous systems cannot be directly transferred to the Synapt XS; this tutorial provides an overview of modifiable settings. We focus on the application of Synapt XS HRMS for untargeted metabolomics of polar and non-polar metabolites across diverse biological matrices. However, the procedures described for sample preparation, chromatographic separation, and data analysis are largely transferable to other high-performance devices. The tutorial includes step-by-step protocols for sample preparation, chromatographic separation of polar and non-polar compounds, adjustment of TWIMS parameters for labile metabolites, and the use of Progenesis QI, including creation of in-house spectral libraries and integration of existing fragment databases. Additionally, strategies for data acquisition modes and initial quality control and preprocessing are presented and discussed. Together, these methods provide a framework for highly sensitive, reproducible metabolomics analyses and, apart from MS-specific parameters, are broadly applicable to modern mass spectrometers from various manufacturers with comparable performance.

Keywords:  High-resolution mass spectrometry; Ion heating; LC-MS protocols; Synapt XS; Traveling wave ion mobility; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.chroma.2026.466991
Anal Chem. 2026 Apr 21.

METLIN 960 K: An Empirical Tandem Mass Spectrometry Data Resource.

Winnie Uritboonthai, Aries E Aisporna, Linh Hoang, Corey Hoang, Elizabeth M Billings, Gary Siuzdak.

METLIN 960 K represents the largest collection of experimentally acquired small-molecule MS/MS spectra currently available. We introduce a reengineered publicaly accessible METLIN platform integrating high-resolution tandem mass spectrometry (MS/MS) data for over 960,000 empirically validated molecular standards. This scale was enabled by a high-throughput experimental framework integrating acoustic droplet ejection with high-throughput LC-MS/MS acquisition, allowing systematic empirical generation of MS/MS spectra from authentic standards. In addition to scale, METLIN 960 K provides a uniquely standardized MS/MS data set, with spectra acquired under controlled and consistent conditions across ionization modes and collision energies, enabling reproducible spectral comparison and machine-learning applications. Each compound is characterized by MS/MS spectra acquired in both positive and negative ionization modes across four collision energies (0, 10, 20, and 40 eV), enabling comprehensive fragmentation coverage and improved structural annotation. Designed as a reference library for XCMS-METLIN and compatible with machine-learning workflows, METLIN 960 K supports high-fidelity spectral matching, neutral loss analysis, and filtering of misannotations, including annotation of in-source fragments and biologically synchronized ranking of candidate metabolites. The platform also provides empirically derived MRM transitions on all standards (via METLIN-MRM), supporting quantitative method development across a chemically diverse range of metabolites, natural products, lipids, peptides, pharmaceuticals, and toxicants. A redesigned interface enables efficient querying by exact mass, formula, or structure with direct access to curated spectra and metadata. Two additional resources enhance identification: (1) METLIN Core, a high-frequency-use subset for rapid searching, and (2) > 1.02 million additional structures without MS/MS data for hypothesis generation. Derived exclusively from authentic standards, METLIN 960 K (https://metlin.scripps.edu) provides the largest publicly available empirical MS/MS database, delivering high-confidence annotation for both untargeted and targeted mass spectrometry workflows.

DOI: https://doi.org/10.1021/acs.analchem.5c08031
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 19. pii: S1570-0232(26)00172-8. [Epub ahead of print]1277 125083

Development and validation of a green DLLME-LC-MS/MS method for apixaban quantification in human plasma using Box-Behnken design optimization with application to pharmacokinetic studies and sustainability assessment.

Ahmed Serag, Manal E Alosaimi, Maram H Abduljabbar, Adnan Alharbi, Faisal Alsenani, Farooq M Almutairi, Majed A Algarni, Reem M Alnemari, Atiah H Almalki.

  A novel dispersive liquid-liquid microextraction coupled with liquid chromatography-tandem mass spectrometry method (DLLME-LC-MS/MS) was developed and validated for apixaban quantification in human plasma. Box-Behnken experimental design was employed to systematically optimize DLLME parameters including disperser solvent volume (acetonitrile, 500-1500 μL), extraction solvent volume (ethyl acetate, 100-250 μL), sample pH (3-9), and centrifugation time (3-9 min). The optimized DLLME conditions achieved 97.97% extraction recovery with excellent precision, consuming only 1.5 mL total organic solvents per sample and representing significant solvent reduction compared to conventional extraction methods. Chromatographic separation was achieved on a Poroshell 120 EC-C18 column with 5-min isocratic elution using positive electrospray ionization tandem mass spectrometry detection. The method demonstrated excellent analytical performance with lower limit of quantification of 1 ng/mL, wide linear range of 1-1000 ng/mL, and comprehensive validation meeting ICH M10 guidelines for selectivity, accuracy, precision, recovery, matrix effects, and stability. Clinical applicability was confirmed through pharmacokinetic studies in healthy volunteers following single 5 mg apixaban administration, with pharmacokinetic parameters (Cmax 107.12 ng/mL, tmax 4 h, t½ 11.16 h, AUC0→∞ 1706.82 ng·h/mL) demonstrating excellent agreement with published literature values. Multi-metric greenness assessment yielded favorable scores: MoGAPI 70%, CaFRI 72/100, BAGI 75.0/100, and WAC RGB 12 whiteness 80.1%, confirming superior environmental sustainability and practical applicability. The developed method provides a robust, sensitive and green analytical platform suitable for therapeutic drug monitoring, bioequivalence studies, and pharmacokinetic investigations of apixaban.

Keywords:  Apixaban; Box-Behnken design; Dispersive liquid-liquid microextraction; Green analytical chemistry; LC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2026.125083
J Mass Spectrom. 2026 May;61(5): e70059

A Simple, Rapid, and Clinically Transferable LC-MS/MS Method for the Detection of Sex Steroid Hormone Metabolic Profiles.

Yongjuan Jia, Xingli Liu, Yongmei Chen, Wei Li.

  Comprehensive profiling of sex steroid hormones (SSHs) is of great significance for elucidating the pathogenesis of SSHs-related diseases. However, it is challenging because of the low endogenous abundance and the presence of multiple analytes with similar structures and isomers. In this study, we developed a simple, rapid, and clinically transferable method for the profiling of 28 SSHs in human serum samples. After liquid-liquid extraction, the serum samples were directly detected by liquid chromatography-tandem mass spectrometry. Twenty-three isomers or structural analogues were separated on Kinetex F5 column within 14 min. Good linearities were achieved in the quantitative range for each analyte with the regression coefficients R2 > 0.99088. The limits of quantitation of the method were in the range from 0.005 to 1 ng/mL. The recoveries were in the range of 90.4%-107.9% with the intraday and interday precisions within 14.35%. This method had been successfully applied for the determination of SSHs in human serum samples with simple operation and good accuracy and could provide a viable approach for clinical screening of SSHs.

Keywords:  liquid chromatography–tandem mass spectrometry (LC–MS/MS); metabolites; serum; sex steroid hormones

DOI:  https://doi.org/10.1002/jms.70059
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 19. pii: S1570-0232(26)00171-6. [Epub ahead of print]1277 125082

A rapid UPLC-MS/MS method for therapeutic drug monitoring of bupivacaine: Validation and clinical application in perioperative analgesia.

Chang Wang, Shijiao Zhang, Wenqing Liu, Zizhuo Shao, Aijia Cao, Chen Jia, Xia Song, Haisheng Jiao, Yinliang Bai.

   BACKGROUND: Bupivacaine (BPC) is a cornerstone of perioperative analgesia, yet its narrow therapeutic index and high interindividual pharmacokinetic variability complicate dosing and increase toxicity risks. A robust, high-throughput analytical platform is needed to support therapeutic drug monitoring (TDM) and individualized therapy.
METHODS: A rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantifying BPC in human plasma was developed and fully validated according to FDA and Chinese Pharmacopoeia guidelines. Sample preparation consisted of a simple protein precipitation step using acetonitrile containing clozapine as the internal standard. Chromatographic separation was achieved within 2.5 min on a C18 column using gradient elution, enabling high-throughput analysis suitable for routine TDM.
RESULTS: The method demonstrated excellent linearity from 1.0 to 500.0 μg/L (R2 > 0.995) and a short run time of 2.5 min, allowing batch analysis of up to 50 samples within 3 h. Intra- and inter-day precision (coefficient of variation) were < 15%, and accuracy ranged between 85% and 115% across quality control levels. The lower limit of quantification was 1.0 μg/L. Extraction recovery was consistent (90.9%-94.5%) and matrix effects were negligible. In a pilot clinical study involving 40 patients, the method successfully differentiated the pharmacokinetic profiles of two formulations: conventional BPC (75 mg) showed rapid distribution and elimination, while liposomal BPC (133 mg) maintained sustained plasma concentrations over 24 h.
CONCLUSION: This validated UPLC-MS/MS method is a rapid, sensitive, and high-throughput tool for BPC TDM that represents a promising candidate for implementation in clinical laboratories following appropriate local validation. Its successful clinical application highlights its utility in characterizing formulation-dependent pharmacokinetics and could assist in the development of precision dosing in perioperative analgesia to optimize efficacy and safety.

Keywords:  Bupivacaine; Liposomal bupivacaine; Method validation; Perioperative analgesia; Therapeutic drug monitoring; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2026.125082
ACS Omega. 2026 Apr 14. 11(14): 21878-21889

Confident Structural Annotation of Bile Acid Isomers Combining Electron-Activated Dissociation or Ion Mobility with High-Resolution Mass Spectrometry in Metabolomics.

Thomas A Brunet, Rémy De Boni, Justine Massias, Luiz F M Rodrigues, Guillaume Rossignol, Arnaud Salvador, Yohann Clément, Carlos A Sorgi, Sophie Ayciriex.

Accurate identification of lipid isomers remains a major challenge in metabolomics, particularly for bile acids (BAs), whose biological functions critically depend on the number, position, and stereochemistry of hydroxyl groups as well as on the nature of their conjugation. Conventional tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) often fails to resolve isomeric lipids differing only in subtle structural features, leading to an underestimation of lipidome complexity. Here, we present an analytical framework integrating electron-activated dissociation (EAD) or ion mobility spectrometry (IM) with high-resolution liquid chromatography-mass spectrometry to improve annotation of BA isomers in untargeted metabolomics. EAD provides nonergodic fragmentation that preserves labile bonds and reveals site-specific details, while IM separates ions by their collision cross section (CCS), supplying orthogonal structural information. Using synthetic standards and human plasma extracts, we show that EAD-derived diagnostic ions markedly enhance structural discrimination and enable consistent and reproducible identification of BA isomers compared to CID-only workflows, while CCS measurements alone lack sufficient robustness to reliably distinguish closely related BA isomers across measurements. The integrated data set enables confident assignment of positional and conjugation variants, contributing to a more complete representation of BA diversity. This multidimensional approach offers a robust platform for structural lipidomics and supports the establishment of transferable spectral and mobility libraries to advance metabolome annotation accuracy.

DOI: https://doi.org/10.1021/acsomega.5c12361
Nat Commun. 2026 Apr 20.

Structure-informed deep generation enables de novo metabolite annotation in untargeted metabolomics.

Hongmiao Wang, Haosong Zhang, Zheng-Jiang Zhu.

Metabolite annotation, especially the discovery of unknown metabolites, remains a fundamental challenge in mass spectrometry-based untargeted metabolomics due to limited reference mass spectra. Here we present MetGenX, a structure-informed encoder-decoder neural network that enables efficient and controllable generation of metabolite structures directly from MS2 spectra. By reformulating the spectrum-to-structure task as a structure-to-structure generation problem, MetGenX significantly improves generation accuracy and chemical space coverage. In independent tests, it achieved top-1 accuracy of 55.9% on 1388 NIST MS2 spectra and 68.5% on 1681 spectra from real biological samples, outperforming existing in silico tools. Its structure-informed design ensures robust performance across both positive and negative ionization modes without retraining. Applying a multi-step annotation workflow to mouse liver untargeted metabolomics data, MetGenX identified two previously uncharacterized metabolites absent from major human metabolome databases. These results demonstrate MetGenX's strong potential to advance de novo metabolite annotation and facilitate the discovery of uncharacterized chemical entities.

DOI: https://doi.org/10.1038/s41467-026-72149-6
Anal Bioanal Chem. 2026 Apr 21.

Miniaturized chromatography-based lipidomics methods towards single-cell analysis.

Klidel Fae Rellin, Michael Witting.

  Lipidomics provides detailed insight into lipid metabolism and cellular function, but conventional workflows typically rely on bulk samples that mask cellular heterogeneity. Advances in analytical chemistry are enabling lipid analysis from extremely limited material, driving the development of miniaturized chromatography-mass spectrometry (LC-MS) workflows for low-input and single-cell studies. This review summarizes recent progress in miniaturized chromatography-based lipidomics. Reducing chromatographic scale improves ionization efficiency, sensitivity, and separation performance while minimizing sample consumption. We discuss key enabling technologies, such as low-flow electrospray interfaces and the integration of ion mobility (IM) spectrometry as an orthogonal separation dimension for lipid identification. Methodological considerations for low-input lipidomics are also addressed, particularly sample preparation and quantitative challenges at picogram-scale analyte levels. Finally, we highlight future directions in automation, microfluidics, and multidimensional separations. Together, these developments position miniaturized chromatography as a critical platform for advancing single-cell lipidomics and high-resolution studies of lipid metabolism.

Keywords:  Ion mobility; Lipidomics; Liquid chromatography; Mass spectrometry; NanoLC; Single cell lipidomics

DOI:  https://doi.org/10.1007/s00216-026-06488-0
Drug Des Devel Ther. 2026 ;20 585730

Targeted Comprehensive Screening of Neuropsychiatric Drugs in Serum by LC-MS/MS: Method Development and Three-Year Clinical Utility.

Eunbin Chong, Se-Eun Koo, Sang-Mi Kim, Miryung Chun, Eun Yeon Joo, Hong Jin Jeon, Soo-Youn Lee.

   Background: The increasing prevalence of neuropsychiatric medication misuse highlights the critical need for efficient drug screening in clinical laboratories. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) application for broad serum drug screening remains limited in many clinical settings. Serum analysis is particularly valuable for identifying current drug exposure, confirming the cause of intoxication, and monitoring patient medication adherence.
Purpose: This study aimed to develop and validate an LC-MS/MS method using multiple reaction monitoring followed by information-dependent acquisition-triggered enhanced product ion scans for the simultaneous qualitative identification of 77 neuropsychiatric drugs, and to present its large-scale clinical application over three years.
Methods: Validation included precision, accuracy, recovery, matrix effects, selectivity, and interference. A minimal serum volume (20 μL) underwent simple protein precipitation, with preparation time under 15 minutes per batch. This method was applied to 1,021 clinical serum specimens.
Results: Chromatographic separation was completed in 15 minutes. The precisions (CV) for calculated concentrations and retention times were < 30% and < 1.5%, respectively. Accuracy was assessed for 60 of the 77 analytes (100% agreement). Mean recovery and matrix effects were 93.1% and 99.9%, respectively. Most test orders originated from psychiatry and neurology outpatients. Although fewer orders came from the Department of Emergency Medicine (EM), the EM patients exhibited a higher positivity rate. The most frequently detected drug classes were benzodiazepines (37.9%), antidepressants (30.7%), and antipsychotics (19.7%). Common specific analytes included clonazepam/7-aminoclonazepam (10.2%) and lorazepam (10.0%).
Conclusion: We developed and validated a LC-MS/MS method for the simultaneous detection of a wide range of neuropsychiatric drugs. The method proved essential for verifying medication history, identifying non-adherence, and diagnosing acute intoxication. Our findings highlight the critical role of broad-spectrum serum screening in managing medication-related emergencies and optimizing long-term therapeutic care.

Keywords:  antidepressants; antipsychotics; benzodiazepines; drug screening; mass spectrometry; serum

DOI:  https://doi.org/10.2147/DDDT.S585730
Anal Bioanal Chem. 2026 Apr 23.

A polarity-based two-dimensional LC-MS/MS distribution strategy combined with ion identity molecular networking for enhanced metabolite annotation in Astragalus Radix species.

Sijian Chen, Xiaoshuang Li, Shuiyuan Yang, Danshu Shi, Yingyuan Lu, Pengfei Tu.

  A polarity-based two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) distribution strategy was established by integrating hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) within a heart-cutting configuration. The resulting LC-MS data were further processed using an ion identity molecular networking (IIMN)-based annotation workflow, which systematically consolidates adducts, in-source fragments, and isotopes across positive and negative ionization modes, thereby facilitating coherent molecular network construction and structural inference. When applied to metabolomic profiling of two Astragalus Radix (AR) species, the proposed strategy enabled the characterization of 253 metabolites spanning multiple chemical classes and the identification of 45 discriminative metabolites associated with botanical origin. In addition, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) revealed distinct tissue-specific distribution patterns of representative differential flavonoids, with predominant localization in the phloem and xylem, providing complementary spatial evidence for species differentiation. Overall, the polarity-based 2DLC-MS/MS distribution strategy with IIMN workflow offers enhanced separation efficiency, improves annotation confidence, and supports structural interpretation of structurally related metabolites. This strategy provides a practical and extensible approach for in-depth characterization of complex plant metabolomes and quality evaluation of natural products.

Keywords:  Astragali Radix; Ion identity molecular networking; Metabolite characterization; Polarity-based two-dimensional liquid chromatography

DOI:  https://doi.org/10.1007/s00216-026-06499-x
J Sep Sci. 2026 Apr;49(4): e70403

An Accurate Method Based on Ultra-High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry for the Quantification of Five Cannabinoids in Juvenile and Adult Caenorhabditis elegans.

Estatira Sepehr, Yang Zhao, Cory A Vaught, Jessica A Camacho, Bonnie Welch, Jeffrey Yourick, Robert L Sprando, Piper Reid Hunt.

  Caenorhabditis elegans is an alternative model organism for toxicology research that aligns with the 3Rs principle (Replace, Reduce, Refine) and new approaches to modernize toxicity testing strategies. This study developed and single-laboratory validated a sensitive ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) method for quantifying five cannabinoids- cannabidivarin, cannabigerol, cannabidiol, cannabinol, and cannabichromene- in juvenile and adult C. elegans matrices. Homogenized C. elegans samples were spiked with labeled internal standards and subjected to protein precipitation. The resulting supernatants were injected onto a Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 × 100 mm), coupled to an Agilent 6460 Triple Quadrupole mass spectrometer detector, allowing for the detection and quantification of target analytes in a total run time of 14 min using a 50 µL sample volume. Single-laboratory method validation was performed using spiked quality control samples, consisting of six replicates at four concentrations of all cannabinoids in juvenile and adult C. elegans matrices, analyzed over three consecutive days. The validated method demonstrated linear regression calibration curves with R2 ≥ 0.99 across the concentration range of 0.1-7.5 µg/mL for all cannabinoids. The intra-day accuracy in juvenile and adult C. elegans was within 85-108% and 97-109% of the fortified concentration, respectively, with intra-day precision between 1.28-4.62% relative standard deviation (RSD) and 0.64-5.60% RSD. Similarly, the inter-day accuracy was within 86-110% and 97-106% of the fortified concentration, respectively, with the inter-day precision between 2.06-5.62% RSD and 1.42-10.9% RSD. This single laboratory validated UHPLC-ESI-MS/MS method provides accurate and reliable cannabinoid quantification and facilitates the translational utility of cannabinoid toxicity testing in C. elegans. Analytical method development to accurately detect chemicals within diverse tissue matrices supports dosimetry and toxicokinetic analyses, essential steps toward investigating food chemical safety.

Keywords:  Caenorhabditis elegans; UHPLC‐ESI‐MS/MS; cannabinoids; method development; single‐laboratory validation

DOI:  https://doi.org/10.1002/jssc.70403
Drug Test Anal. 2026 Apr 22.

A Novel Workflow for Fast Elucidation of Drug Metabolites for Screening-Combining In Silico Metabolite Prediction With Trapped Ion Mobility QTOF-MS.

Annette Zschiesche, Birgit Schneider, Ilona Nordhorn, Carsten Baessmann, Laura M Huppertz, Jürgen Kempf.

  Urine is one of the preferred matrices for standard toxicological analysis, which makes the inclusion of drug metabolites in targeted and untargeted screening mandatory. Mass spectrometry is key for substance identification, but updating methods for emerging substances like new psychoactive substances (NPS) is challenging due to the limited availability of reference standards for metabolites. This is particularly problematic for drugs that are barely or not detectable at all in urine. Insufficient metabolic knowledge and lack of spectral data carry the risk of false negatives. This study evaluates a non-targeted workflow using ultrahigh-performance liquid chromatography-trapped ion mobility spectrometry time-of-flight mass spectrometry (UHPLC-timsTOF-MS) and dedicated processing software (MetaboScape), integrating in silico metabolite prediction (BioTransformer), fragmentation (MetFrag), collision cross-section (CCS) prediction, and library searching. Quetiapine was selected as a model compound. Phase I metabolites were generated via pooled human liver microsomes (pHLMs) and analyzed by UHPLC-timsTOF-MS. Features were extracted and annotated with MetaboScape. The workflow successfully annotated 20 phase I metabolites in the pHLM assay, with 13 confirmed by library matching and 18 by BioTransformer. These metabolites were added to a targeted UHPLC-QTOF-MS method for analysis of 30 quetiapine-positive ante- and post-mortem urine samples from forensic casework. This revealed N-, O-dealkyl and carboxylated metabolites as the most abundant biomarkers in human urine. This integrated approach enables rapid and reliable metabolite detection, supports biomarker discovery, and facilitates routine screening updates, especially for substances without reference standards. Although not intended for exhaustive metabolic characterization, it offers practical applicability in evolving drug landscapes.

Keywords:  CCS; PASEF; biomarkers; in silico prediction; trapped ion mobility spectrometry

DOI:  https://doi.org/10.1002/dta.70062
Anal Chem. 2026 Apr 22.

FlashMRM: An Automated Platform for MRM Method Generation and Iterative Optimization Based on High-Resolution Spectra.

Zecang You, Ao Guo, Changzhi Shi, Jiahu Deng, Jingzhi Yao, Jia Tu, Junjie Yang, Shipei Xing, Mingliang Fang.

High-throughput targeted analysis in exposomics relies on multiple reaction monitoring (MRM) using liquid chromatography-triple quadrupole mass spectrometry (LC-TQMS), yet its method development remains limited by dependence on chemical standards, labor-intensive parameter generation, and complex matrix interferences (INTF). Here, we introduce FlashMRM, a web-based platform for the automated generation and optimization of MRM parameters by leveraging high-resolution mass spectrometry (HRMS) databases and experimentally preacquired biosample HRMS data. The pseudotranslation from the large-scale HRMS database to the TQMS database (TQDB) was achieved through mass unit normalization, retention time prediction, and cross-instrument collision energy conversion algorithms. FlashMRM integrated Pseudo TQDB containing over 25,000 candidate analytes and INTF TQDB, which encompasses ∼44,000 potential interference MS features in biological matrices, utilizing a dual-weighted scoring model to balance the sensitivity and specificity. Leveraging a library of pesticides with highly diverse chemical structures as a representative model, FlashMRM generated transitions for 255 pesticides with sensitivity comparable to experimentally developed MRM transitions while increasing the specificity score from 0.60 to 0.68. At a spiked concentration of 10 ng/L in urine, the number of Top 5 detectable transitions increased from 194 in experiments to 233 using FlashMRM with optimized specificity weighting, enhancing trace-level detection under biomatrix interference. In the analysis of human urine samples, FlashMRM achieved high-confidence TQMS detection for 55 of 99 targeted exposure biomarkers. FlashMRM enables sensitive and accurate large-scale targeted screening on TQMS without standards and extensive preanalysis injections, integrating optimized scoring algorithms and built-in databases within a user-friendly web platform.

DOI: https://doi.org/10.1021/acs.analchem.6c00179
Anal Methods. 2026 Apr 21.

A green analytical method for identification and quantification of carcinogenic impurity N-nitroso-desformyl riociguat in riociguat formulations by UHPLC-MS/MS.

Veeraswami Boddu, Manoj Kumar Mandapaka.

Riociguat, a vasodilatory drug for treating pulmonary hypertension (PH), might generate a nitrosamine drug substance-related impurity (NDSRI), reported as N-nitroso-desformyl riociguat (NNDFR). NDSRIs are a class of nitrosamine impurities that are specific to each drug and are structurally related to the active pharmaceutical ingredient (API) either or both sharing a common molecular backbone but differing by specific functional groups. Such impurities have drawn growing regulatory attention due to their recognized genotoxic and carcinogenic potential, in which the identification and quantification in the product is mandatory. Thus, a sensitive and reliable Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method is developed employing a Shimpack-GIS CN column (150 mm × 4.6 mm × 3 µm) with a gradient of 0.1% formic acid in water, and 0.1% formic acid in methanol for chromatography separation, and in multiple reaction monitoring (MRM) mode in an electrospray ionization (ESI) environment. Optimized conditions focused on chromatographic selectivity, ionization efficiency, and matrix interference reduction. Method validation for the identification of NNDFR in the riociguat drug product confirmed that this method achieved baseline separation of NNDFR isomers with retention times of 10.037 (Isomer-1) and 10.579 (Isomer-2) minutes, prohibiting interferences from other compounds. Validation also confirmed excellent linearity (R2 > 0.99), precision (RSD < 1.3%), and accuracy (recoveries from 100.5% to 110.4%). The LOD and LOQ were determined to be 0.4 ppm and 1.22 ppm, respectively. A 24-hour stability study confirmed the analyte integrity and method robustness at various flow rates and column temperatures, and under various filtration conditions, ensuring consistent performance. This validated UHPLC-MS/MS method is robust, sensitive, and reliable for routinely quantifying NNDFR in riociguat drug products. Subsequently, the analytical method developed was assessed for environmental sustainability using established greenness assessment tools. The AGREE score was calculated to be 0.73, AGREEprep to be 0.69, and the BAGI value to be 70.0. These values reflected a favorable environmental profile, efficient sample preparation, and practical sustainability. Together, these metrics confirmed the method's suitability for both regulatory compliance and routine application in industrial quality control laboratories.

DOI: https://doi.org/10.1039/d6ay00279j
Clin Chim Acta. 2026 Apr 20. pii: S0009-8981(26)00206-8. [Epub ahead of print] 121024

Evaluation and clinical application of an accurate isotope dilution liquid chromatography-tandem mass spectrometry method for serum glycocholic acid.

Zhonggan Jin, Yuanzhu Ou, Ming Zong, Zhihan Ye, Zhizheng Li, Sujie Zhang, Hewei Sun, Xiaoyu Fan, Xialin Luo, Lingli Jiang, Yi Ju.

   OBJECTIVES: Glycocholic acid (GCA) is an important identified biomarker for various hepatobiliary diseases. Accurate measurement of GCA is essential for diagnosis and treatment of hepatobiliary diseases. Our aim was to develop and validate a high accuracy method for the quantification of GCA in serum.
METHODS: An isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS) method has been developed and validated. The assay validation was performed, including precision, trueness, matrix effects, linearity, interference, and carry-over, etc. Furthermore, the developed method was applied to comparison with three latex-enhanced immunoturbidimetric assays and for external quality assessment (EQA) of clinical systems.
RESULTS: Excellent linearity coefficients (R2 = 0.9998) were obtained in the range from 0.063 to 49.53 μg/g. The lowest limit of detection and limit of quantification was 0.1 ng/mL and 2.3 ng/mL, respectively. The total imprecisions were below 2.33% for low, medium, and high concentration samples. Good recoveries were achieved at three spiked levels (98.94%-101.80%), and a relative expanded uncertainty was <3% (k = 2). No interference, matrix effect, and carry-over were observed. Significant bias across routine GCA assays obtained by immunoassays and ID-LC/MS/MS highlighted the urgency of standardization for the measurement of GCA.
CONCLUSIONS: This study successfully established an accurate quantitative method for detecting serum GCA using ID-LC/MS/MS. The method could serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. It could support EQA management for GCA testing and promote GCA standardization.

Keywords:  Glycocholic acid; Isotope dilution-liquid chromatography-tandem mass spectrometry; SI units; Traceability

DOI:  https://doi.org/10.1016/j.cca.2026.121024
Biomed Chromatogr. 2026 Jun;40(6): e70445

Multidimensional and Hybrid Separation Strategies for Body Fluids and Tissues: Comparative Performance and Translational Considerations.

Uttam Prasad Panigrahy, Yaso Deepika Mamidisetti, Kuchukuntla Mounika, Jainendra Kumar Battineni, Divya Amaravadi, M Akiful Haque.

  Analytical technologies for body fluids and tissues have advanced substantially over the past decade, particularly in chromatographic, electrophoretic and ion-mobility-based separation strategies designed to address matrix complexity and structural isomerism. Improvements in stationary-phase chemistry, multidimensional liquid chromatography, capillary electrophoresis and mobility-integrated mass spectrometry have enhanced peak capacity, structural discrimination, remains the selection and validation of separation strategies capable of minimizing coelution, ion suppression and quantitative variability in heterogeneous matricesremains the selection and validation of separation strategies capable of minimizing coelution, ion suppression and quantitative variability in heterogeneous matrices. This review examines recent progress in multidimensional chromatography, mobility-resolved workflows, miniaturized separation systems and hybrid imaging-separation platforms, with emphasis on comparative performance, practical implementation constraints and translational reproducibility. Applications across plasma, serum, urine, saliva, cerebrospinal fluid (CSF), tissues and spatially resolved analyses are discussed in the context of analyte chemistry and matrix-dependent method selection. Future progress will depend on standardized multidimensional workflows, validated performance metrics, harmonized reporting frameworks and data-driven optimization strategies that support robust clinical translation. By focusing on separation-centred decision-making and quality assurance, this review provides guidance for analytical scientists seeking reliable molecular characterization of complex human biospecimens.

Keywords:  body fluids; chromatography; mass spectrometry; metabolomics; microfluidics; tissues

DOI:  https://doi.org/10.1002/bmc.70445
Int J Anal Chem. 2026 ;2026 9944493

Quantification of the Active Metabolites 4-Methylaminoantipyrine and 4-Aminoantipyrine of Dipyrone From Human Plasma by LC-MS/MS.

Stefanie Schmidt, Harald Ihmsen, Tobias Golditz, Jürgen Schüttler, Andreas Wehrfritz.

  Dipyrone (metamizole) is widely used as a nonopioid analgesic in perioperative and intensive care settings; however, its administration may be associated with severe adverse effects. In this study, we describe an analytical assay for the quantification of the active metabolites 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA) in human plasma using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). In addition, the protein-unbound fractions of both metabolites were determined by ultrafiltration. Chromatographic separation was performed on a UPLC system using gradient elution, followed by MS/MS detection with an electrospray ionization source. The limits of detection for both metabolites were 100 ng/mL. Across the investigated concentration range (100-10,000 ng/mL), the relative error (%RE) ranged from -6.3% to +3.5%. Intra-day and inter-assay variability were below 10%. Method validation was conducted in accordance with the 2018 FDA Bioanalytical Method Validation Guidance, and all evaluated parameters met the required criteria for analytical accuracy and precision. The developed assay is suitable for monitoring dipyrone metabolites and may support the prevention of potential overdosing during prolonged analgesic therapy or in intensive care settings.

Keywords:  4-aminoantipyrine; 4-methylaminoantipyrine; dipyrone; liquid chromatography; mass spectrometry

DOI:  https://doi.org/10.1155/ianc/9944493
J Pharm Biomed Anal. 2026 Apr 12. pii: S0731-7085(26)00182-2. [Epub ahead of print]277 117514

Development and validation of an UHPLC-MS/MS method for simultaneous quantification of Tazobactam, Meropenem, Vancomycin, Linezolid and Piperacillin in human plasma: Application in critically ill patients.

Jacopo Lucchetti, Claudia Fracasso, Davide Cesana, Francesca Dore, Stefano Finazzi, Marco Gobbi.

  Antibiotic treatment for severe infections in critically ill patients might be challenging. The pathophysiological changes occurring in these patients can alter the pharmacokinetic profiles of antibiotics, resulting in ineffective or incorrect treatments, with potentially dangerous consequences. In this scenario, therapeutic drug monitoring is crucial for timely adjusting therapy and improving antibiotic effectiveness. In this study, we developed and validated an ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry method for simultaneous quantification of five antibiotics - Tazobactam, Meropenem, Vancomycin, Linezolid and Piperacillin - in human plasma. Detection was achieved using a triple-quadrupole mass spectrometer in multiple reaction monitoring mode, with chromatographic separation via a binary gradient elution in 10 min. Method validation following ICH M10 guidelines confirmed that a single-step protein precipitation efficiently extracted all five drugs, ensuring accurate and precise quantification without matrix interference. The method was then successfully applied to measure antibiotics concentrations in plasma from 64 critically ill patients admitted in intensive care unit and treated with one or more of these drugs simultaneously.

Keywords:  Antibiotics; Human plasma; Intensive care unit; Protein precipitation; Therapeutic drug monitoring; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2026.117514
Talanta. 2026 Apr 16. pii: S0039-9140(26)00415-7. [Epub ahead of print]308 129759

Evolving LC-MS hyphenations: strengths, limitations, and emerging trends in bioanalytical applications.

Bhavna P Kapse, Rishabh P Soni.

  Hyphenated analytical techniques, particularly liquid chromatography-mass spectrometry (LC-MS), play a central role in addressing the increasing complexity of modern bioanalytical challenges. Recent advancements in LC-MS hyphenation strategies have significantly improved sample preparation efficiency, analyte enrichment, and overall analytical performance. The integration of ion mobility spectrometry provides an additional orthogonal separation dimension, particularly effective for drug metabolism research and isomer discrimination. Furthermore, capillary electrophoresis hyphenation enables effective analysis of polar and charged metabolites, while ambient ionization approaches such as DESI and DART support rapid screening with minimal sample preparation. Tandem setups involving UV, fluorescence, and NMR provide essential layers of structural confirmation. Additionally, the shift toward microfluidic lab-on-a-chip platforms enables miniaturization, thereby reducing sample volume requirements and increasing analytical throughput. Despite these advances, challenges related to instrumental complexity, data interpretation, and regulatory harmonization continue to limit broader translation into routine practice. Collectively, these developments highlight the transformative potential of LC-MS while also emphasizing critical gaps that will shape future bioanalytical advancements.

Keywords:  Capillary electrophoresis–mass spectrometry (CE–MS); Ion mobility spectrometry (IMS); Liquid chromatography–mass spectrometry (LC–MS); Tandem mass spectrometry (MS/MS)

DOI:  https://doi.org/10.1016/j.talanta.2026.129759
Anal Chem. 2026 Apr 21.

CASMDB: An Open-Source Database of Metabolite Annotation Data for 1D 1H NMR-Based Metabolomics.

Morgan W Hayward, Luca G Mureddu, Gary S Thompson, Marie M Phelan, Edward J Brooksbank, Geerten W Vuister.

In metabolomics analyses, databases are invaluable for the identification of individual metabolites in experimentally collected samples. Publicly available databases for NMR-based metabolomics are unfortunately incomplete with respect to experimental conditions, such as pH, temperature, and NMR field strength, which all affect the observed signals. Moreover, derived NMR annotation parameters, such as peak positions and multiplet patterns, are also often incomplete and contain crucial errors. Hence, these databases are often inadequate for the analyses of experimental samples across a range of conditions, most notably field strength. In this paper, we describe the collection, remediation, and integration of annotation data from the publicly available HMDB, BRMB, and GISSMO NMR metabolomics databases to build the CcpNmr Analysis Simulated Metabolomics Database (CASMDB). CASMDB contains 1932 unique and fully annotated metabolite entries that allow for accurate simulation of spectra at arbitrary field strengths. CASMDB can be downloaded as a standalone, versioned repository from GitHub and easily augmented with new entries. CASMDB underpins the visualizing of experimental and simulated metabolite references and allows for 1D 1H NMR-based metabolomics studies.

DOI: https://doi.org/10.1021/acs.analchem.5c04525
Microbiome Res Rep. 2026 ;5(1): 7

Development of a multi-targeted metabolomics platform for semi-quantification of faecal metabolites: a proof-of-concept analysis in human faeces.

Kayo Ikuta, Akihiro Kunisawa, Itaru Dekio, Arisa Ito, Qiuyi Wang, Kazuhiro Kawamura, Masaki Yamada, Sumi Nakamura, Yoshihiro Hayakawa, Takuma Higurashi, Junko Iida, Eiichiro Fukusaki, Toru Suzuki, Mitsuharu Matsumoto.

  Objectives: Targeted metabolomic analysis of faecal samples has been limited by narrow chemical coverage. Here, we established a multiplexed, triple quadrupole mass spectrometry (TQMS)-based targeted metabolomics workflow. This workflow allows accurate detection and semi-quantification of diverse faecal metabolites and provides a methodological platform for studying host-microbiome metabolic interactions. Methods: Faecal metabolomes from germ-free (GF) mice, ex-germ-free (Ex-GF) mice, and human participants were analysed using TQMS-based targeted metabolomics. The analysis comprised multiple methods targeting amino acids and their derivatives, carbohydrates, short-chain fatty acids, bile acids, lipid mediators, and phospholipids. Results: In total, 607 low-molecular-weight metabolites in 44 chemical categories were detected and semi-quantified. Faecal metabolomes of GF and Ex-GF mice were analysed, uncovering 341 intestinal microbiome-dependent metabolites. A proof-of-concept analysis using faecal samples from five patients with colorectal cancer demonstrated the successful application of this platform to human clinical material, highlighting its strong potential for future disease-oriented metabolomic investigations. Conclusion: We developed a multi-targeted faecal metabolomics platform that substantially expands the chemical space accessible to targeted analysis. This workflow provides a methodological foundation for future large-scale and translational studies.

Keywords:  Intestinal microbiome; faeces; germ-free mice; metabolome; triple quadrupole mass spectrometry

DOI:  https://doi.org/10.20517/mrr.2025.85
Metabolomics. 2026 Apr 23. pii: 56. [Epub ahead of print]22(3):

Metabolic and lipidomic stability in EDTA plasma from hospitalized patients: impact of refrigerated storage time and additional freeze-thaw cycle.

Sander Johannes Thorbjørnsen Guttorm, Liana Gukasyan, Christian Prebensen, Niclas G Karlsson, Aleksander Rygh Holten, Dag Kvale, Helge Rootwelt, Katja Benedikte Prestø Elgstøen, Erik Koldberg Amundsen.

   BACKGROUND: Global metabolomics and lipidomics are increasingly applied in clinical research. Storage variability can compromise analyte stability and omics data quality, especially in clinical settings. While previous studies have focused on healthy individuals, the stability of the metabolome and lipidome in patient samples remains underexplored.
OBJECTIVE: To identify metabolites and lipids most susceptible to be affected by post-centrifugation storage time and a single additional freeze-thaw cycle in EDTA plasma from hospitalized patients.
METHODS: EDTA plasma samples from 20 patients acutely hospitalized in a medical ward were collected (K2EDTA, 5 mL, with gel) and stored at 4 °C for 0, 24, and 72 h post-centrifugation. All samples underwent one additional freeze-thaw cycle and were analyzed using global liquid chromatography-mass spectrometry (LC-MS) metabolomics and lipidomics.
RESULTS: Approximately 90% of the global metabolome and lipidome remained stable and robust to the pre-analytical factors induced. Metabolomic profiles showed a storage time-dependent increase in differentially abundant features, while most lipidomic alterations occurred within the first 24 h. A total of 116 annotated compounds exceeded a Cohen's d effect size threshold of ± 0.25, including lactate, hypoxanthine, oxoproline, fatty acid(20:4), and lysophosphatidylcholine(16:0).
CONCLUSION: The plasma metabolome and lipidome are largely robust to common storage conditions in patient samples. However, refrigerated plasma storage time post-centrifugation and an additional freeze-thaw cycle can induce biologically significant changes in specific metabolites and lipids. This study is among the first to evaluate metabolomic and lipidomic stability using clinical samples and our data supports a sample collection that can be easily implemented in clinical laboratory workflows.

Keywords:  EDTA plasma; Lipidome; Metabolome; Storage stability; Storage time

DOI:  https://doi.org/10.1007/s11306-026-02428-2
J Nat Prod. 2026 Apr 23.

Curation of Mass Spectrometry Reference Data for Improved Identification and Dereplication of Cyanobacterial Specialized Metabolites.

Franziska Schanbacher, Anne Dax, Michael A Stravs, Timo H J Niedermeyer, Elisabeth M-L Janssen.

High-resolution tandem mass spectrometry (HRMS/MS) is a powerful tool for screening organic compounds in complex samples. A critical step in identifying candidate structures is the comparison of sample HRMS/MS spectra with those of reference spectral libraries. The effectiveness of this spectral matching hinges on two key factors: (i) how well the library's content aligns with the suspect compound list and (ii) the quality and diversity of the reference spectra for each compound. Yet the scarcity of natural product reference materials on the market often necessitates non-targeted analysis. In this study, we systematically acquired and curated HRMS/MS reference spectra for specialized metabolites from cyanobacteria, which are vastly underrepresented in the current libraries. Previously, MassBank EU included spectra for only 14 such compounds. We have significantly expanded the publicly available data, contributing 2905 unique spectra representing 150 distinct cyanobacterial metabolites. A proof-of-concept analysis demonstrates up to 5-fold increased annotation success and revealed shortcomings in current libraries, underscoring the need for continued data enrichment. In particular, future efforts should prioritize the inclusion of HRMS/MS spectra for diverse adduct ions to improve identification confidence and broaden the analytical coverage.

DOI: https://doi.org/10.1021/acs.jnatprod.6c00107
ACS Meas Sci Au. 2026 Apr 15. 6(2): 311-323

Dark Reactions in Microdroplets Explain Widespread Artifacts in Metabolomic Profiling.

Xiaowei Song, Jinheng Xu, Chenglong Sun, Lecheng Lyu, Hongqian Kui, Ruiping Zhang, Zeper Abliz, Richard N Zare.

  Reliable ion annotation and identification remain persistent challenges in mass-spectrometry-based untargeted metabolomics. Here, we elucidate the identities, sources, and formation mechanisms of numerous previously unexplained ions by showing that microdroplets formed during electrospray ionization can promote a wide array of chemical transformations. These include redox, addition, condensation, reductive amination, decarboxylative coupling, and radical reactions, many of which are facilitated at the gas-aqueous interface by reactive oxygen and nitrogen species. Activation of metabolite chemical bonds generates cations, anions, and radical intermediates through the loss of protons, electrons, or functional groups, ultimately leading to bond formation and the generation of artifactual ions or false positive ions. These artifacts are frequently misassigned as endogenous metabolites and account for hundreds of thousands of previously unidentified features. As an example, we show that in a recently published untargeted metabolomics analysis of 1969 ions, the annotation rate was substantially improved to over 50% from the previous value of 9%, showing the importance of this new form of identification. Finally, we describe practical strategies to minimize artifactual ion formation, thereby improving the reliability of metabolomic analyses.

Keywords:  dark metabolomics; electrospray ionization; interfacial chemistry; mass spectrometry; water microdroplet

DOI:  https://doi.org/10.1021/acsmeasuresciau.5c00146
J Chromatogr A. 2026 Apr 15. pii: S0021-9673(26)00326-2. [Epub ahead of print]1778 466996

Automated solid-phase extraction coupled with heart-cutting two‑dimensional liquid chromatography for simultaneous quantification of thirteen fat-soluble vitamins in foods for special medical purposes.

Lulu Sun, Qianqian Li, Shuai Zhu, Qiqi Sun, Shijian Zheng, Tingting Feng, Huadong Sheng, Shuifeng Zhang.

  A method based on heart‑cutting two‑dimensional liquid chromatography coupled with automated solid‑phase extraction was developed and validated for the simultaneous determination of thirteen fat‑soluble vitamins, including vitamins A, D₂, D₃, four tocopherols (α‑, β‑, γ‑, δ‑tocopherol), α‑tocotrienol, four β‑carotene isomers (all‑trans‑, 9‑cis‑, 13‑cis‑, 15‑cis‑β‑carotene), and lutein-in foods for special medical purposes (FSMP). The method integrates automated SPE sample purification with a two‑dimensional chromatographic separation that employs a C30 column in the first dimension and a PFP column in the second dimension. A heart‑cutting valve‑switching strategy was implemented to selectively transfer the vitamin‑D‑containing fraction from the first to the second dimension, thereby overcoming matrix interference and enabling accurate trace‑level quantification. The method demonstrated excellent performance: limits of detection (LODs) ranged from 0.01 μg/L (vitamin A) to 0.05 mg/L (tocopherols and α‑tocotrienol) and 0.001-0.005 mg/L (β‑carotene isomers), with corresponding limits of quantification (LOQs) from 0.03 μg/L to 0.15-0.015 mg/L. Spike recoveries for all analytes were between 88.5% and 108.2%, with intra‑ and inter‑day precision (RSD) below 5.0%. When applied to commercial FSMP samples, results showed good agreement (relative deviations < 10%) with established Chinese national standard methods. This sensitive, robust, and highly automated 2D‑LC‑SPE platform provides a reliable, high‑throughput solution for quality control, nutrition‑label compliance verification, and regulatory monitoring of FSMP products.

Keywords:  Automated solid-phase extraction (SPE); Fat-soluble vitamins; Foods for special medical purposes (FSMP); Method validation; Two-dimensional liquid chromatography; UV detector

DOI:  https://doi.org/10.1016/j.chroma.2026.466996
Drug Test Anal. 2026 Apr 23.

A Comparison of Three Analytical Procedures for Oxandrolone Long-Term Metabolite Determination in Urine Samples.

B Stojanovic, S Pfeffer, G Gmeiner.

  In contemporary antidoping analysis, doping control laboratories are confronted with a wide range of evolving analytical challenges. Continuous methodological refinement is required in this dynamic field, particularly with respect to analytical strategies for the detection and confirmation of prohibited substances. The present study focuses on the comparative evaluation of three analytical strategies for the determination of long-term metabolites (LTMs) of the anabolic androgenic steroid (AAS) oxandrolone (Oxa) in human urine. The identification and implementation of LTMs in routine doping control have substantially enhanced the detection capabilities of antidoping laboratories, leading to an increased number of adverse analytical findings, particularly for AAS compounds. The primary objective of this work was to develop and validate novel confirmatory analytical procedures for Oxa LTMs and to compare their performance with that of an already established method. The investigated approaches comprise the following: (1) gas chromatography-mass spectrometry (GC-MS) following enzymatic hydrolysis of phase II metabolites and subsequent silylation, (2) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) of methylated phase II metabolites, and (3) LC-HRMS analysis of hydrolyzed phase II metabolites. The first two approaches are reported here for the first time in the context of LTM analysis in antidoping applications. These methodologies represent innovative analytical strategies that may be transferable to other target analytes and thereby contribute to further improvements in sports drug testing.

Keywords:  GC‐MS; LC‐HRMS; derivatization; oxandrolone long term metabolites; sport drug testing

DOI:  https://doi.org/10.1002/dta.70080
Anal Chim Acta. 2026 Jul 01. pii: S0003-2670(26)00421-6. [Epub ahead of print]1405 345471

3D-printed removable-cap device with embedded magnet for magnetic solid phase extraction.

Camilla Fonseca Silva, Matheus Martins Guedes, Daiane Dulcileia Moraes de Paula, Keyller Bastos Borges.

   BACKGROUND: Magnetic adsorbents are widely employed in sample preparation, but magnetic solid-phase extraction (MSPE) still faces limitations related to magnet handling and pipetting, which increase time, variability, and operational costs. To overcome these drawbacks, we developed a 3D-printed magnetic removable-cap extraction device aimed at simplifying manipulation, improving reproducibility, and minimizing solvent consumption in the extraction step. As a proof of concept, a restricted-double-access magnetic polypyrrole adsorbent was synthesized, characterized, and applied to the extraction of nifedipine (NDP) and nimodipine (NMDP) from human plasma prior to chromatographic analysis.
RESULTS: The magnetic adsorbent was evaluated using the 3D-printed magnetic removable-cap device and compared with conventional MSPE approaches. Structural and morphological characterization confirmed its mesoporous and hydrophobic properties, together with strong macromolecule exclusion. After optimization, both extraction formats demonstrated suitable precision and accuracy, while the 3D-printed device provided a faster workflow and simpler operation. Optimal conditions involved 15 mg of adsorbent, 250 μL of plasma at pH 10.0, 1000 μL of methanol for elution, and 60 s of agitation. The HPLC-UV method showed linearity from 100 to 3500 ng mL-1, with adequate selectivity and accuracy. The limits of detection and quantitation were 50 and 100 ng mL-1, respectively, and protein removal reached 93%. The magnetic adsorbent was reusable for up to three extraction cycles without loss of performance.
SIGNIFICANCE: Coupling the 3D-printed device with the magnetic adsorbent enabled efficient, selective, and reproducible determination of NDP and NMDP in human plasma while minimizing sample handling and solvent use. Green analytical metrics confirmed this workflow as an eco-friendly, rapid, and reliable alternative for bioanalytical sample preparation.

Keywords:  Calcium channel blockers; Human plasma; Magnetic adsorbent; Magnetic removable cap extraction; Polypyrrole; Restricted access material

DOI:  https://doi.org/10.1016/j.aca.2026.345471
Anal Chem. 2026 Apr 21.

A Universal Framework for Blood Ionome Extraction and Intelligent Quality Control in 1H NMR Metabolomics.

Anastasios Theodorou, Ivan Vučković, Eirini Papadimitriou, Konstantinos Papakonstantinou, Vasiliki Taki, Alexandra Louka, Costas Papaloukas, Constantine D Stalikas, Nikolaos Giormezis, Fotini Paliogianni, Ian R Lanza, Justin Stebbing, Panteleimon G Takis.

Nuclear magnetic resonance (NMR) spectroscopy is a cornerstone of metabolomics and clinical bioanalysis; yet, its routine clinical adoption is limited by challenges in quality control (QC) and spectral interpretation. In blood-based NMR studies, ethylenediaminetetraacetic acid (EDTA) and its metal complexes are a major source of spectral interference, obscuring endogenous metabolites while increasing the analytical complexity. Here, we introduce a fully automated cheminformatics platform that enables EDTA-aware QC and the quantitative extraction of metal ion information while preserving metabolic integrity. Using large plasma and serum cohorts acquired at 500 and 600 MHz, we establish a multicenter-validated workflow for accurate quantification of calcium (Ca2+), magnesium (Mg2+), and zinc (Zn2+) directly from routine 1H NMR spectra (R2 > 0.9 versus clinical and spectrometric assays). The platform harmonizes data sets across instruments and cohorts through automated baseline reconstruction. Application to a large breast cancer cohort reveals a stage-dependent decline in circulating Zn2+, consistent with disrupted zinc homeostasis in invasive disease. This work bridges metabolomics and metallomics, advancing the translational utility of NMR in precision medicine.

DOI: https://doi.org/10.1021/acs.analchem.5c08255
Rapid Commun Mass Spectrom. 2026 Jul 30. 40(14): e70091

Detection of Microbiological Contamination and Malfunctions in Technical Equipment Used in the Milk Processing Industry Using Acyl-Homoserine Lactones With Liquid Chromatography/Triple Quadrupole Mass Spectrometry.

S Koch, A L Kertsch, S Baldermann, L Gorzki.

   RATIONALE: Bacterial contamination causes sensory changes and health risks and is a critical quality factor in the food industry. Plate-based microbiological methods have long incubation times, making rapid contamination control difficult. Homoserine lactones (HSLs) from Gram-negative bacteria mediate quorum sensing that drives biofilm and toxin formation; our approach uses four homoserine lactones to rapidly identify contamination.
METHODS: A sample preparation and LC/MS/MS method were established and validated for the four HSLs of different polarity: N-butyryl-L-homoserine lactone, N-(3-hydroxy-butanoyl)-L-homoserine lactone, N-(3-oxooctanoyl)-L-homoserine lactone, and N-(3-oxododecanoyl)-L-homoserine lactone. This method uses positive electron spray ionization with a triple quadrupole mass analyzer and γ-decalactone as the internal standard. The validated method was then used to analyze HSLs extracted from technical cooling water by solid-phase extraction.
RESULTS: The method provides same-day results without production shutdowns and baseline-separated chromatography. Applied to real cases, HSLs profiling identified contamination and malfunctions with six true-positive and six true-negative classifications.
CONCLUSION: HSL-targeted LC/MS/MS offers a practical, rapid, and sensitive tool for detecting leaks, biofilms, and hidden microbial activity in complex industrial water matrices. The approach enables earlier risk management, reduced downtime, and routine monitoring in milk-processing facilities.

Keywords:  LC/MS/MS; acyl‐homoserine lactones; biofilm detection; biomarkers; quorum sensing

DOI:  https://doi.org/10.1002/rcm.70091
J Sep Sci. 2026 Apr;49(4): e70419

Heart-Cutting Two-Dimensional Liquid Chromatography-Isotope Ratio Mass Spectrometry for Compound-Specific δ13C Analysis of Water-Soluble B Vitamins in Complex Supplement Matrices.

Sarah P Rockel, Jacqueline Martiny, Maik A Jochmann, Torsten C Schmidt.

  Compound-specific stable carbon isotope analysis by one-dimensional liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is fundamentally constrained by the requirement for fully aqueous mobile phases, which limits chromatographic selectivity and prevents effective separation of analytes from complex sample matrices. Here, a heart-cutting two-dimensional LC-IRMS method (2D-LC-IRMS) is presented for the first compound-specific δ13C analysis of the water-soluble vitamins B5 (pantothenic acid) and B9 (folic acid) in commercial dietary supplements and fortified beverages. An organic-modified reversed-phase separation in the first dimension achieved matrix reduction, while a fully aqueous second-dimension separation ensured IRMS compatibility and delivered baseline-resolved analyte peaks for precise isotope determination. Validation demonstrated linear calibration over 2-100 mgC L-1 with R2 ≥ 0.9999, repeatability of ≤ 0.13‰, and isotope-stability-based method detection limits of 5 mgC L-1. Analyzed δ13C values were independent of chromatographic configuration, confirming that heart-cutting transfer does not introduce isotope fractionation. Application to 12 commercial products, including tablets, effervescent formulations, powdered supplements, and an energy drink, yielded δ13C ranges of -20.6‰ to -32.9‰ for vitamin B5 and -20.4‰ to -36.4‰ for vitamin B9, reflecting differences in synthetic production routes across manufacturers. The presented workflow extends LC-IRMS to the compound class of water soluble vitamins that were so far inaccessible to this technique and provides a broadly applicable strategy for compound-specific isotope analysis of polar analytes in challenging matrices.

Keywords:  dietary supplements; food authenticity; isotope ratio mass spectrometry; reversed‐phase chromatography; stable carbon isotope analysis

DOI:  https://doi.org/10.1002/jssc.70419
J Am Soc Mass Spectrom. 2026 Apr 24.

Direct Comparison of MRM-MS and PRM-MS Methods for Quantitative Ganglioside Analysis.

Akeem Sanni, Abderrahmane Koraich, Judith Nwaiwu, Arvin Saffarian Delkhosh, Yehia Mechref.

  Gangliosides are structurally diverse, low-abundance glycosphingolipids central to neuronal signaling and cancer progression; however, their quantitative analysis is hindered by low ionization efficiency, structural heterogeneity, and complex isomeric patterns. Targeted mass spectrometry (MS) represents a powerful approach for resolving these challenges, yet systematic comparisons of multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) for native gangliosides remain limited. Here, we developed and optimized a targeted LC-MS/MS workflow to directly evaluate PRM on a Q-Exactive HF Orbitrap versus MRM on a TSQ Vantage triple quadrupole. Collision-energy optimization revealed distinct fragmentation behaviors across platforms, identifying optimal normalized collision energies (NCE) for PRM of 28 for GD1a and 25 for GD2, GT1b, GM1, and GQ1b, whereas optimal CE values for MRM were 35 for GD1a, GD2, and GT1b, and 30 for GQ1b. Additionally, PRM enabled multiplexing up to 15 transitions per analyte, improving signal-to-noise up to ∼4-fold and reducing %RSD through postacquisition transition summation. High-energy collision dissociation (HCD) used in PRM generated a richer array of fragment ions, including informative cross-ring cleavages and low-mass diagnostic ions, providing superior structural confidence compared to CID fragmentation in MRM. Notably, PRM uniquely enabled quantification of GM1, which exceeded the mass range of the triple quadrupole instrument. Applied to post-mortem human brain tissue extracts, PRM distinguished GD1a and GD1b isomers with high specificity. These findings establish PRM as a robust, highly sensitive, and structurally informative platform for comprehensive ganglioside profiling in complex biological matrices.

Keywords:  CID; HCD; LC-MS/MS; MRM; PRM; gangliosides

DOI:  https://doi.org/10.1021/jasms.6c00071
J Am Soc Mass Spectrom. 2026 Apr 22.

CorrelandSW: Correlation Networks from LC-MS Data.

Andrea Kosinova, Jiri Gruz.

  The increasing size and complexity of mass spectrometry (MS) data sets necessitate advanced computational tools. This study presents Correland, a MATLAB-based software for clustering and visualizing metabolite correlations through weighted correlation networks, which directly represent pairwise associations. Its effectiveness was tested on a data set from nontargeted LC-MS analysis of 14-day-old Arabidopsis thaliana seedlings inoculated with Alternaria alternata and Fusarium oxysporum, demonstrating effective clustering of biosynthetically related metabolites. The ion grouping algorithm resulted in a substantial reduction in network scale (83 nodes/metabolites from approximately 900 features). In addition to network construction, Correland enables pseudomolecular ion identification with a success rate of 86-90% achieved in Arabidopsis extract. Network density is reduced by limiting visible edges, producing interpretable and visually coherent networks generated in a single step using rescaled Pearson correlation coefficients.

Keywords:  LC-MS data; correlation; metabolomics; network

DOI:  https://doi.org/10.1021/jasms.6c00065
Talanta. 2026 Apr 15. pii: S0039-9140(26)00425-X. [Epub ahead of print]308 129769

Solvent-assisted sensing ability of 19F reporter in simultaneous discrimination and quantification of proline and 4-hydroxyproline isomers.

Li-Hua Xu, Yu-Kai Wang, Ya-Ting Chen, Xun-Cheng Su.

  Quantitative analysis of metabolites with very similar chemical structures in complex biological matrices remains challenging. Although 19F nuclear magnetic resonance (NMR) spectroscopy offers high sensitivity and minimal background interference in analytical chemistry, limitations in quantification of analytes with highly similar structures still remain. Here, we report a solvent-assisted 19F chemical shift separation strategy that enables direct distinction and quantification of multiple analytes with similar chemical structures, for example, proline (Pro) and 4-hydroxyproline (4-Hyp) isomers. We found that mono-fluorinated tags exhibit substantially greater sensitivity to solvent polarity than trifluoromethyl-containing tags, enabling finer modulation of chemical shifts and improved spectral resolution of 19F-NMR signals. Simultaneous discrimination and quantification of Pro and 4-Hyp isomers were achieved by 19F NMR in the presence of a certain amount of organic solvent in aqueous solution. This method was directly applied to in-situ quantification of Pro and 4-Hyp isomers in commercially available fetal bovine serum (FBS), human plasma and gelatin samples, demonstrating solvent modulation is an effective approach for enhanced chemical shift sensing of important analytes with very similar chemical structures in complex biological environments using 19F NMR.

Keywords:  (19)F-tag; Biofluid; Hydroxyproline; NMR; Proline

DOI:  https://doi.org/10.1016/j.talanta.2026.129769
Anal Sci Adv. 2026 Jun;7 e70082

Dry Deposition Strategies for MALDI-MS Imaging: Principles, Advances and Emerging Applications.

Toufik Mahamdi, Oscar Yanes.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has become a pivotal tool in biomedical research, enabling untargeted, spatially resolved analysis of molecular species in complex biological tissues. Among the critical steps in MALDI-MSI workflows, matrix deposition significantly influences sensitivity, spatial resolution and reproducibility by affecting crystal morphology and analyte extraction efficiency. While wet deposition techniques, such as manual spray-coating, electrospray deposition and automated systems, have been widely adopted, they often suffer from limitations related to solvent-induced analyte delocalisation. These challenges have driven increasing interest in dry matrix deposition methods, which aim to eliminate or minimise solvent use to enhance spatial fidelity and image quality. This review summarises the main dry deposition strategies used in MALDI-MSI, covering their principles, instrumentation, benefits and limitations. It also discusses recent comparative studies (2023-2025) between wet and dry approaches, examining their impact on analytical performance and the increasing importance of dry deposition in high-resolution spatial omics.

DOI: https://doi.org/10.1002/ansa.70082