bims-metlip 2026-04-05 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–04–05
thirty papers selected by
Sofia Costa, Matterworks

LC-MS/MS methods for synthetic opioids in urine: analytical and clinical evaluation.
Multiplexed Tandem Mass Spectrometry Imaging Enables Large-Scale Isomer Mapping and Annotation in Tissues.
1H-qNMR analysis of human urine: Validation of an external standard approach for absolute metabolite quantitation.
Facile one-pot cleavage with in-situ generated aluminum iodide for reliable quantification of alkylphenol ethoxylates in waterborne coatings.
[Determination of 3-nitropropionic acid in plasma by high-performance liquid chromatography-tandem mass spectrometry method].
Reduction of Matrix Effects in Liquid Chromatography-Tandem Mass Spectrometry by a Dilution Approach for the Analysis of 245 Pesticide Residues in Selected Vegetables.
Artificial Intelligence-Based LC-OzESI-MRM for Isomer-Resolved Triacylglycerol Profiling by In-Source Ozonolysis.
Development of a Specialized Method for Simultaneous Quantification of Functional Intestinal Metabolites by GC/MS-Based Metabolomics.
Simultaneous Determination of ABA, IAA, SA, and JA by UPLC-ESI-MS/MS.
LC-MS Analysis and Quantification of Plants Apocarotenoids.
Simultaneous quantitation of free testosterone and free thyroid hormones in human serum by equilibrium dialysis LC-MS/MS.
Characterization and Quantification of Strigolactones in Root Exudates.
High-throughput quantification of tacrolimus in whole blood by acoustic ejection mass spectrometry: analytical validation and comparative assessment against conventional LC-MS/MS for therapeutic drug monitoring.
Single-Injection Multi-Omics Analysis by Direct Infusion Mass Spectrometry.
Implementing Annotation Confidence Scoring in Untargeted Mass Spectrometry Workflows for Small Molecule Analysis.
Extraction and Analysis of Cytokinins from Mammalian Tissues by HPLC-HRMS.
N-aminophthalimide-derived unsaturated PC reaction-assisted analysis of carbon-carbon double bond position and cis/trans isomerism in fatty acid chains based on UHPLC-ESI-MS/MS.
Evaluation and optimization of SPE pre-processing combined with V-ITEX-GC-MS versus direct V-ITEX-GC-MS for urinary volatilome profiling.
An integrated derivatization-LC-MS strategy reveals cell-type-specific chiral amino acid profiles.
Integrated Single-Cell Mass Spectrometry Imaging and Immunofluorescence Microscopy for Multimodal Characterization of Human Immune Cells.
Spatial Lipidomics Reveals Region-Specific Lipid Remodeling in Scn2a-Deficient Mouse Brain.
Simultaneous Quantification of Salicylic Acid, Pipecolic Acid, and N-Hydroxypipecolic Acid in Plants Using High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry.
Development and validation of an LC-MS/MS method for the analysis of total ascorbic acid capacity in plasma.
Meeting the analytical challenges of fatty-acid engineering: a comprehensive method for triacylglycerol identification and quantitation in omega-3 enhanced oilseeds.
Improved Methods for Recording Accurate Collision Cross Sections Using Cyclic Ion Mobility-Mass Spectrometry.
Assessment of the inaccuracy associated with the quantification of phenolic compounds using UHPLC-DAD and HPLC-MS/MS.
Achieving Single-Cell Resolution via Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) on Different Platforms.
Ultra-Performance Liquid Chromatography-Electrospray Ionization-Ion Trap Tandem Mass Spectrometry for Fragmentation of Cyclic Dipeptides: Methodology and Application to the Chemical Profiling of the Fungus Hormonema dematioides.
Analysis of ultra-short-chain and short-chain PFAS in environmental samples: A review.
Combining intact and oxidative LC-MS/MS methods to mitigate health risks from Ostreopsis cf. ovata.

Clin Chim Acta. 2026 Mar 31. pii: S0009-8981(26)00173-7. [Epub ahead of print] 120991

LC-MS/MS methods for synthetic opioids in urine: analytical and clinical evaluation.

Selen Al, Aykut Kul, Olcay Sagirli.

  Opioids are potent analgesic compounds that exert their effects on the central nervous system and are widely used in clinical practice; however, their increasing misuse and associated health risks necessitate reliable and sensitive analytical monitoring. Urine remains the primary biological matrix for assessing compliance and detecting illicit opioid use. This review critically evaluates liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of buprenorphine, methadone, tramadol, fentanyl, and pethidine in urine. Across the reviewed studies, diverse sample preparation strategies were employed, with calibration ranges typically reported at the ng/mL level. Limits of detection ranged from 0.005 to 10 ng/mL, while limits of quantification varied between 0.025 and 208 ng/mL. Analysis times ranged between approximately 2.5 and 24 min, reflecting differences in analytical throughput and chromatographic resolution. Sample preparation strategies, including solid-phase extraction, liquid-liquid extraction, and microextraction techniques, were critically compared in terms of recovery, matrix effects, and efficiency. Chromatographic separation predominantly relied on C18 columns with mobile phases containing formic acid, ammonium formate, or ammonium bicarbonate, and all methods applied positive electrospray ionization. Importantly, analytical trade-offs between opioid-specific assays and broader multi-analyte LC-MS/MS workflows were identified, particularly regarding sensitivity, metabolite coverage, and robustness against matrix effects. Overall, LC-MS/MS remains the most reliable confirmatory approach for urinary opioid analysis; however, challenges persist in the harmonization of analytical protocols, differentiation of structurally similar analogs, and standardization of matrix-effect evaluation. Future developments should focus on harmonization and integration of advanced analytical strategies to enhance clinical and forensic applicability.

Keywords:  Forensic toxicology; Opioids, LC-MS/MS, urine; Synthetic opioids

DOI:  https://doi.org/10.1016/j.cca.2026.120991
Angew Chem Int Ed Engl. 2026 Mar 30. e22119

Multiplexed Tandem Mass Spectrometry Imaging Enables Large-Scale Isomer Mapping and Annotation in Tissues.

Varun V Sharma, Gabor Toth, Robert Martinis, Cathrin E Hansen, Gijs Kooij, Ingela Lanekoff.

  Accurate molecular annotation is essential for deciphering biochemical processes in spatial biology. Here, we present a scalable and broadly applicable molecular annotation tool for tandem mass spectrometry imaging (MS2I). Our workflow includes parallel image acquisition (PIA) for parallel MS2I and an open-access computational framework for spatial similarity networking (SSN) that enables molecular annotation of MS2I data with isomeric specificity. The PIA enables simultaneous untargeted MSI and targeted MS2I ensuring structure-specific imaging of hundreds of molecules in a single experiment. The SSN increases annotation confidence through graph-based spatial correlation of product ion distributions, opening up new avenues for data investigation and annotation from both MSI and MS2I data. By integrating PIA and SSN into a single workflow, we visualize and annotate 134 phospholipid isomers and isobars in mouse brain tissue. Furthermore, we demonstrate the biological utility of the platform by mapping cholesterol metabolism in human multiple sclerosis brain tissue, revealing lesion-associated cholesterol oxidation pathways. Finally, we propose annotation confidence levels for structural annotation in MSI. Overall, PIA and SSN together provide large-scale, structure-specific MSI, expanding the scope for spatial metabolomics, lipidomics, and chemical pathology through molecular annotation beyond current capabilities.

Keywords:  annotation; isomers; lipids; mass spectrometry imaging; multiple sclerosis

DOI:  https://doi.org/10.1002/anie.202522119
Anal Biochem. 2026 Mar 31. pii: S0003-2697(26)00077-1. [Epub ahead of print] 116121

1H-qNMR analysis of human urine: Validation of an external standard approach for absolute metabolite quantitation.

Eduardo Zul-Hernández, Noemí Waksman-Minsky, Verónica M Rivas-Galindo, Paula Cordero-Pérez, Ernesto Sánchez-Mendoza, J Ricardo Lucio-Gutiérrez, Alma L Saucedo.

  Quantitative nuclear magnetic resonance (qNMR) spectroscopy is a powerful tool for metabolite analysis, offering reproducibility, minimal sample preparation, and non-destructive measurements. In this study, a 1H-qNMR method employing external standard calibration was developed and validated for the absolute quantification of 14 representative metabolites in human urine. Metabolite selection was based on their consistent occurrence in the Human Urine Metabolome and supported by literature frequency, resulting in a chemically diverse set including amino acids, organic acids, carbohydrates, and amines. The method was validated according to EUROLAB and ENFSI guidelines, assessing specificity, linearity, precision, accuracy, sensitivity, robustness, and matrix effects. The results demonstrated excellent analytical performance: calibration curves were highly linear (R2 ≥ 0.9987), accuracy and precision met acceptance criteria (% error < 10%, %RSD < 15%), and recoveries ranged from 87% to 103%. Limits of detection and quantification were suitable for physiological urine concentrations, while matrix effects remained within the accepted ±20% range, except for one analyte. Robustness testing confirmed method stability under minor variations, with only aromatic resonances requiring control of relaxation delays. Application to urine samples from healthy individuals confirmed the suitability of the approach for quantifying both low- and high-abundance metabolites. Importantly, the inclusion of structurally diverse metabolites enabled different quantification strategies, such as spectral deconvolution in crowded regions and signal summation for conformers. Overall, the validated method provides a robust and flexible platform for absolute metabolite quantification in urine by 1H-qNMR. This framework enhances the reliability of NMR-based metabolomics and supports its application in clinical research.

Keywords:  NMR; absolute quantification; external standard calibration; metabolomics; method validation

DOI:  https://doi.org/10.1016/j.ab.2026.116121
Talanta. 2026 Mar 27. pii: S0039-9140(26)00388-7. [Epub ahead of print]306 129732

Facile one-pot cleavage with in-situ generated aluminum iodide for reliable quantification of alkylphenol ethoxylates in waterborne coatings.

He-Wei Ma, Cai-Dan Zhang, Jian-Xun Luo, Yu-Dan Yi.

  Alkylphenol ethoxylates (APEOs) in waterborne coatings (WBCs) pose substantial health and environmental risks due to their estrogenic degradation metabolites, necessitating robust analytical methods to meet stringent regulations. Conventional liquid chromatography-mass spectrometry (LC-MS) methods often suffer from congener separation difficulties and severe matrix interference. To overcome these limitations, we developed an analytical procedure based on facile one-pot cleavage technique using aluminum iodine (AlI3) generated in-situ for the reliable quantification of APEOs in WBCs. The method integrates three core innovations: (1) rapid dehydration with MgCl2 (1.5 × the mass of sample water) to prevent moisture-induced AlI3 decomposition; (2) efficient, one-pot AlI3-mediated cleavage under ultrasonic irradiation (85 °C, 20 min) for quantitative APEOs-to-alkylphenol (AP) conversion; and (3) simple, quantitative AP isolation via tert-butyl methyl ether (BME) extraction. Validated using gas chromatography-mass spectrometry (GC-MS), the method exhibited excellent linearity (R2 > 0.999), low limits of quantification (3.0 mg/kg for OPEO10, 9.0 mg/kg for NPEO10), satisfactory recoveries (92.9-107.4%) and relative standard deviations (RSDs) ≤ 11.8%. Compared to routine LC-MS methods, this approach effectively mitigates matrix effects, reduces analytical cost, and offers a greener alternative suitable for batch analysis. Application to 20 real WBC samples confirmed its reliability in quantifying APEOs across concentration ranges, from low-level contaminants to high-load formulations. Overall, this integrated strategy enables sensitive, rapid and accurate APEO analysis, presenting a promising new avenue for screening these contaminants in complex sample matrices such as WBCs.

Keywords:  AlI(3)-Mediated cleavage; MgCl(2)-Based dehydration; Nonylphenol ethoxylates; Octylphenol ethoxylates

DOI:  https://doi.org/10.1016/j.talanta.2026.129732
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2026 Mar 20. 44(3): 232-234

[Determination of 3-nitropropionic acid in plasma by high-performance liquid chromatography-tandem mass spectrometry method].

H L Li, R C Liu, B Zhou.

  Objective: To establish a rapid method for detecting 3-nitropropionic acid (3-NPA) in plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) . Methods: In May 2024, the plasma samples were extracted by precipitating proteins with methanol∶ acetonitrile (volume ratio 1∶1). Then, they were analyzed using a C18 chromatographic column with a gradient elution using a 0.1% formic acid aqueous solution and acetonitrile as the mobile phase, in the electrospray negative ion mode (ESI-) and the multiple reaction monitoring mode (MRM). The method was quantified using external standards. The linearity range, sensitivity, accuracy, and precision of the method were verified. Results: The HPLC-MS/MS method for measuring 3-NPA in plasma exhibited a good linear relationship within the concentration range of 1-50 μg/L, with a correlation coefficient of 0.9996. The detection limit of the method was 0.1 μg/L, and the quantitative lower limit was 0.3 μg/L. The recovery rate of the spiked samples ranged from 92.0% to 103.4%, and the intra-and inter-day precisions were 2.4%-9.1% and 2.8%-7.3%, respectively. Conclusion: The HPLC-MS/MS method is simple, rapid, sensitive and accurate, and is suitable for the qualitative and quantitative analysis of 3-NPA in the plasma of patients with sugarcane poisoning or occupational exposed population.

Keywords:  3-nitropropionic acid; High-performance liquid chromatography-tandem mass spectrometry; Plasma; Precipitation protein method

DOI:  https://doi.org/10.3760/cma.j.cn121094-20241105-00503
J Sep Sci. 2026 Apr;49(4): e70401

Reduction of Matrix Effects in Liquid Chromatography-Tandem Mass Spectrometry by a Dilution Approach for the Analysis of 245 Pesticide Residues in Selected Vegetables.

Yeong-Ju Jo, Jeong Yoon Choi, Syed Wasim Sardar, Abd Elaziz Sulieman Ahmed Ishag.

  Matrix effects in liquid chromatography-tandem mass spectrometry remain a significant challenge in pesticide residue analysis. Ion suppression can introduce uncertainty in quantification and adversely affect both precision and accuracy. To address this, sample dilution is an effective mitigation approach. In this study, matrix effects of 245 compounds (pesticides and metabolites) were evaluated across six representative vegetable matrices: garlic, onion, chives, perilla leaf, cucumber, and tomato, under various dilution factors. Results showed that increasing the dilution factor consistently reduced matrix effects in most matrices. For instance, high-effect matrices like garlic and onion, a 10-fold dilution approximately doubled the number of compounds within the "weak" matrix effect range. In contrast, perilla leaves and chives, which had moderate matrix effects, required two-fold and five-fold dilutions, respectively, to eliminate compounds in the "strong" effect range. On the other hand, cucumbers and tomatoes exhibited minimal matrix effects, as over 95% of their compounds were already in the weak range before dilution, so further dilution had a limited impact. Overall, these results demonstrated that dilution substantially reduced matrix effects, particularly in complex matrices. Specifically, optimal dilution factors of tenfold for garlic, onion, perilla leaf, and chives, and five-fold for cucumber and tomato, enabled accurate quantification using solvent-based calibration curves. In cases where matrix effects could not be reduced, the use of stable isotope-labeled internal standards or other matrix mitigation strategies can serve as an effective alternative.

Keywords:  dilution; liquid chromatography; mass spectrometry; matrix effect; pesticides

DOI:  https://doi.org/10.1002/jssc.70401
Anal Chem. 2026 Mar 31.

Artificial Intelligence-Based LC-OzESI-MRM for Isomer-Resolved Triacylglycerol Profiling by In-Source Ozonolysis.

Caitlin E Randolph, Sanjay Iyer, Connor Beveridge, Stephen J Blanksby, Berwyck L J Poad, Shane Tichy, Amy C Clingenpeel, Gaurav Chopra.

The structural complexity of triacylglycerols (TGs) and the positional specificity of carbon-carbon double bonds (C═C) within fatty acyl (FA) chains present a formidable challenge in structural lipidomics. Traditional tandem mass spectrometry (MS) approaches often lack the capability to distinguish TG isomers, limiting insights into lipid biochemistry and isomer-specific functionality. Here, we introduce an online liquid chromatography (LC) approach that enables isomer-specific TG identification through in-source ozonolysis via ozone electrospray ionization (OzESI) combined with multiple-reaction monitoring (MRM) on a triple quadrupole mass spectrometer. By directly incorporating ozone into the electrospray ionization source, our approach generates diagnostic ozonolysis product ions that localize C═C positions without the need for post-ionization reaction chambers or major instrument modifications. We demonstrate the utility of this method using synthetic TG standards and apply it to real-world samples, including the analysis of canola oil across processing stages. To overcome the challenges of data analytics, automated data processing is achieved via CLAW-OzESI-MRM (Comprehensive Lipidomics Automated Workflow for OzESI-MRMs) which includes an artificially intelligent (AI) agentic framework to streamline structural assignment of TGs with C═C specificity. This robust, accessible platform offers enhanced isomeric resolution for lipidomics research, expanding the analytical capabilities of targeted MS workflows in food, biomedical, and industrial applications.

DOI: https://doi.org/10.1021/acs.analchem.5c05709
Gut Microbes Rep. 2024 ;1(1): 2429408

Development of a Specialized Method for Simultaneous Quantification of Functional Intestinal Metabolites by GC/MS-Based Metabolomics.

Kazuki Funahashi, Chol Gyu Lee, Kuniyo Sugitate, Noriko Kagata, Noriko Fukuda, Isaiah Song, Chiharu Ishii, Akiyoshi Hirayama, Shinji Fukuda.

  Intestinal metabolites produced by gut microbes play a significant role in host health. Due to their different chemical structures, they are often analyzed using multiple analyzers and methods, such as gas chromatography/mass spectrometry (GC/MS) for SCFAs and liquid chromatography/mass spectrometry (LC/MS) for bile acids (BAs), amino acids (AAs), and sugars. In this study, we aimed to develop a specialized method for the simultaneous determination of important intestinal metabolites, specifically addressing the main issue of SCFA volatilization during the dry solidification process. We discovered that these compounds can all be measured in fecal samples by GC/MS after trimethylsilyl (TMS) derivatization despite the expected volatility of SCFAs. Validating the results using SCFA standards suggested that the fecal matrix exerts a stabilizing effect. This method enabled the simultaneous quantification of 65 metabolites. For further validation in a biological context, a mouse study showed that high-MAC and high-fat diets increased SCFAs and BAs in feces, respectively, and showed a negative correlation between Alistipes and sugars, all consistent with previous studies. As a result, we successfully developed a specialized simultaneous quantification method for SCFAs, BAs, AAs, AA derivatives, and sugars in fecal samples using GC/MS-based metabolomics in conjunction with a TMS derivatization pretreatment process.

Keywords:  GC/MS; Gut microbiome; TMS derivatization; bile acids; intestinal metabolites; metabolomics; short-chain fatty acids; volatility

DOI:  https://doi.org/10.1080/29933935.2024.2429408
Methods Mol Biol. 2026 ;3026 1-9

Simultaneous Determination of ABA, IAA, SA, and JA by UPLC-ESI-MS/MS.

Carlos de Ollas Valverde, Aurelio Gómez-Cadenas.

  This protocol describes a method for the simultaneous extraction and quantification of the main phytohormones involved in plant stress responses and development-abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA)-from small quantities of plant tissue. It combines minimal extraction and purification steps with high-throughput analysis by ultra-high pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-ESI-MS/MS). Surrogate standards are used for accurate quantification, making the method suitable for diverse plant matrices and absolute quantification.

Keywords:  Abscisic acid; HPLC-ESI-MS/MS; Indole-3-acetic acid; Jasmonic acid; Phytohormones; Plant extracts; Salicylic acid

DOI:  https://doi.org/10.1007/978-1-0716-5214-5_1
Methods Mol Biol. 2026 ;3026 59-68

LC-MS Analysis and Quantification of Plants Apocarotenoids.

Mohamed Salem, Kawthar F Alashoor, Kit Xi Liew, Salim Al-Babili.

  Carotenoid oxidative cleavage products (apocarotenoids; APOs) represent an important class of specialized metabolites that exert diverse and important biological functions. Apocarotenoids are precursors of the plant hormones abscisic acid and strigolactones, pigments such as crocin and picrocrocin, and volatiles such as β-ionone and β-cyclocitral. Recently, carotenoid-derived bioactive metabolites such as zaxinone, anchorene, β-cyclocitral, and retinal have been identified as plant root growth regulators. Analysis of plant APOs is crucial for elucidating their biological function and metabolism as well as for determining their content in food. In this chapter, we describe an efficient protocol for a simultaneous analysis of plant APOs utilizing ultra-high performance liquid chromatographic (UPLC) separation and tandem mass spectrometry (MS). Additionally, we introduce practical details to assist researchers in setting up their experiments, extraction, and analysis of APOs.

Keywords:  Apocarotenoids; Liquid chromatography; Mass spectrometry; Qualitative and quantitative analysis; Rice; Zaxinone

DOI:  https://doi.org/10.1007/978-1-0716-5214-5_5
Anal Chim Acta. 2026 Jun 01. pii: S0003-2670(26)00299-0. [Epub ahead of print]1401 345349

Simultaneous quantitation of free testosterone and free thyroid hormones in human serum by equilibrium dialysis LC-MS/MS.

Alexander Bauer Westbye, Oskar Kelp, Yilu Ye, Marianne Lerbæk Bergmann, Finn Erik Aas, Sandra R Dahl, Per M Thorsby.

BACKGROUND: Testosterone (Te) and the thyroid hormones (THs) thyroxine (T4) and triiodothyronine (T3) are key endocrine hormones regulating development, metabolism and reproductive function. The free (not protein-bound) hormone concentrations more accurately reflect biological activity than total levels. Free hormone measurements are therefore used for clinical decision making in many contexts. Mass spectrometry (MS)-based methods following equilibrium dialysis (ED) are considered "gold standard" for free hormone quantification. Unfortunately, few clinical laboratories offer such measurements today.
RESULTS: A 96-well format ED-LC-MS/MS method incorporating isotope-dilution for simultaneous quantitation of free Te, free T4 and free T3 in clinical samples that used offline (parallel) sample-preparation was successfully developed and validated. The method covered clinically relevant ranges, and clinical samples with a wide concentration range were analyzed to perform a comparison to existing (immunoassay or calculation) methods. Free hormone concentration in 20 healthy blood donors is reported.
SIGNIFICANCE: This method allows direct quantitation of both free testosterone and free thyroid hormones in human samples at clinically relevant concentrations. It can be implemented alongside existing LC-MS/MS methods in specialized clinical laboratories to support improved endocrine assessment and clinical decision making.

DOI: https://doi.org/10.1016/j.aca.2026.345349
Methods Mol Biol. 2026 ;3026 39-58

Characterization and Quantification of Strigolactones in Root Exudates.

Jian You Wang, Guan-Ting Erica Chen, Kawthar F Alashoor, Salim Al-Babili.

  This chapter details the methodology for collecting and processing plant root exudates using a C18 solid-phase extraction column, followed by the isolation, identification, and quantification of strigolactones (SLs) through liquid chromatography-mass spectrometry (LC-MS).

Keywords:  C18 solid-phase extraction column; Liquid chromatography with tandem mass spectrometry (LC-MS/MS); Liquid chromatography-mass spectrometry (LC-MS); Pearl millet; Rhizosphere signals; Rice; Root exudates; Sorghum; Strigolactones

DOI:  https://doi.org/10.1007/978-1-0716-5214-5_4
Clin Chim Acta. 2026 Mar 26. pii: S0009-8981(26)00168-3. [Epub ahead of print]588 120986

High-throughput quantification of tacrolimus in whole blood by acoustic ejection mass spectrometry: analytical validation and comparative assessment against conventional LC-MS/MS for therapeutic drug monitoring.

Xin-Yu Wu, Xiao-Kang Fu, Ao Li, Wei Zhang, Han-Peng Jiang, Na An, Quan-Fei Zhu, Shao-Ting Wang, Yu-Qi Feng.

   BACKGROUND: Therapeutic drug monitoring (TDM) of tacrolimus requires accurate, high-throughput methods, but conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) is limited by lengthy run times and complex sample preparation.
METHODS: We developed a chromatography-free assay for quantification of tacrolimus in whole blood by integrating cold-induced phase separation with acoustic ejection mass spectrometry (AEMS). This workflow only requires less than 10 μL of whole blood, eliminates centrifugation, and enables direct nanoliter-scale injection into the mass spectrometer. The AEMS assay performance was validated in accordance with CLSI C62-A and ICH M10 guidelines using 62 patient samples and matrix-matched calibrators, demonstrating its suitability for high-throughput therapeutic drug monitoring.
RESULTS: The AEMS assay exhibited excellent linearity (0.5-50 μg/L; R2 = 0.9987), with a lower limit of quantification of 0.5 μg/L, which was well below clinically relevant trough concentrations. Accuracy (93.3%-100.1%) and precision (intra-day RSD: 3.7%-11.3%; inter-day RSD: 2.3%-12.4%) met regulatory acceptance criteria. Results showed strong concordance with routine LC-MS/MS (Spearman ρ = 0.972; Passing-Bablok slope = 1.100 [95% CI: 1.026-1.177]). The total analysis cycle time was less than 3 s per sample, enabling a throughput exceeding 1000 samples per hour theoretically.
CONCLUSION: This study establishes the first chromatography-free mass spectrometry platform validated for clinical TDM of TAC. By integrating ultra-high throughput capability, minimal sample consumption, and robust analytical performance, the AEMS workflow offers a transformative analytical solution for high-demand clinical environments, including transplant centers and pediatric monitoring settings.

Keywords:  AEMS; High throughput; Tacrolimus; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.cca.2026.120986
Angew Chem Int Ed Engl. 2026 Mar 30. e19836

Single-Injection Multi-Omics Analysis by Direct Infusion Mass Spectrometry.

Yuming Jiang, Ivan Salladay-Perez, Amanda Momenzadeh, Jesús Muñoz-Estrada, Uktarsh Tripathi, Anthony J Covarrubias, Jesse G Meyer.

  Combined proteomics, metabolomics, and lipidomics analyses require long liquid chromatography-mass spectrometry (LC-MS) run times, limiting throughput and increasing costs for large-scale studies. Here, we present single-injection multi-omics analysis by direct infusion (SMAD), an integrated platform leveraging ion mobility mass spectrometry and self-developed software tools to enable single injection multi-omics analysis without liquid chromatography. SMAD allows quantification of over 9000 metabolite m/z features and over 1300 proteins from the same sample in less than 5 min. We validated the efficiency and reliability of SMAD with three case studies: (1) mouse macrophages after M1/M2 polarization and senescence, (2) a pilot drug screen in human cells, and (3) large-scale high-throughput drug screening of mammalian cells in 96-well plates. Finally, relationships between proteomic and metabolomic data are discovered by machine learning and validated.

Keywords:  chemical biology; lipidomics; macrophages; metabolomics; proteomics

DOI:  https://doi.org/10.1002/anie.202519836
Anal Chem. 2026 Apr 01.

Implementing Annotation Confidence Scoring in Untargeted Mass Spectrometry Workflows for Small Molecule Analysis.

Daniel Krakko, Ralf Tautenhahn, Whitney L Stutts.

Untargeted small molecule analysis by high-resolution mass spectrometry is integral to environmental and biological research, enabling comprehensive characterization of complex samples. However, data interpretation and reporting remain challenging due to the complexity and high dimensionality of molecular features in untargeted data sets. Current data analysis platforms provide integrated tools for processing and annotation yet lack a standardized framework for assigning and reporting annotation confidence. Communicating varying levels of confidence in untargeted data sets continues to pose challenges without an automated ranking system. To address this, a custom scripting node was developed that assigns annotation confidence levels based on the widely adopted Schymanski et al. scoring scheme. While implemented here for metabolomics, the scoring approach is broadly applicable to other untargeted small molecule workflows. The script can be incorporated into a commercial data analysis software package and functions as a standalone postprocessing node, expanding the original five-level system with four new sublevels (levels 3a/3b and 4a/4b) to improve specificity and distinguish cases that fall between established categories. Annotation confidence is assessed using available information from all compound identification workflow nodes (e.g., Predicted Composition, mzVault, mzCloud, ChemSpider), and the consensus scoring algorithm is used to evaluate agreement among search nodes for greater accuracy. Validation using NIST SRM 1950 plasma samples demonstrated 100% scoring accuracy in negative mode and >99.5% in positive mode across both RPLC and HILIC separations. This tool enhances data reporting, improves transparency, and promotes consistency across studies, facilitating standardization and comparability of untargeted metabolomics results.

DOI: https://doi.org/10.1021/acs.analchem.5c03229
Methods Mol Biol. 2026 ;3026 69-77

Extraction and Analysis of Cytokinins from Mammalian Tissues by HPLC-HRMS.

Muhammad Naseem, Kenesha Wilson, Feras Lafi, Elena Bencurova, Khalid Muhammad, Thomas Dandekar, Alfonso Albacete, Eman M Othman.

  Cytokinins (CKs) are adenine-derived plant hormones regulating almost every aspect of plant growth and development. But CK-like compounds may also occur in microbial as well as mammalian systems. Detecting CKs in animal matrices is challenging due to low abundance and complex backgrounds. We describe a high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) protocol for CK extraction, purification, and quantification. Samples are frozen or lyophilized, homogenized, and extracted with methanol: water (80:20, v/v) containing stable isotope labeled CKs as internal standards. Solid-phase extraction (SPE) with C18 cartridges removes proteins and lipids, and purified eluates are dried, reconstituted, and analyzed by reversed-phase HPLC coupled to an Orbitrap mass spectrometer. Accurate-mass detection (≤5 ppm) and diagnostic fragmentation confirm identity, while isotope-dilution calibration enables picomolar sensitivity. This method provides high recovery (>75%) and selectivity, enabling investigation of CK occurrence in ex-planta cellular systems.

Keywords:  Active form; Cytokinins; Non-plant systems; Plant hormones; Signalling molecules; Storage form; Two-component systems

DOI:  https://doi.org/10.1007/978-1-0716-5214-5_6
Anal Chim Acta. 2026 Jun 01. pii: S0003-2670(26)00324-7. [Epub ahead of print]1401 345374

N-aminophthalimide-derived unsaturated PC reaction-assisted analysis of carbon-carbon double bond position and cis/trans isomerism in fatty acid chains based on UHPLC-ESI-MS/MS.

Chunlin Yue, Lingyu Zhao, Jinqi Yang, Yuan Cao, Shan Shu, Yangyang Zhang, Zhenwen Zhao.

   BACKGROUND: Phosphatidylcholines (PCs) are key components of biological membranes and play crucial roles in key physiological processes, where the positions and cis/trans isomerism of carbon-carbon double bonds (CC) in fatty acyl chains critically influence physiological functions and disease mechanisms. The conventional LC-MS technique cannot simultaneously resolve the positional and cis/trans isomers of phosphatidylcholine.
RESULT: To address the limitations of conventional LC-MS/MS in distinguishing PC isomers, we developed a novel method combining N-aminophthalimide (PhthNH2) derivatization with UHPLC-ESI-MS/MS. This approach leverages the specific reaction between PhthNH2 and CC bonds to form N-aziridine adducts, enabling precise localization of double bond positions through characteristic fragment ions while achieving baseline chromatographic separation of cis/trans isomers in monounsaturated PC.
SIGNIFICANCE: A derivatization workflow and data analysis procedure for simultaneously detecting PC CC bonds positional and cis/tans isomers was successfully established and optimized. The method successfully identified 31CC positional isomers in plasma samples and revealed significant differences in PC isomer abundance between colorectal cancer patients and healthy controls, establishing a powerful new tool for investigating PC biology and disease associations.

Keywords:  Aziridination reaction; C=C bonds position and cis/trans isomers; Phosphatidylcholines; UHPLC-ESI-MS/MS

DOI:  https://doi.org/10.1016/j.aca.2026.345374
Anal Bioanal Chem. 2026 Mar 31.

Evaluation and optimization of SPE pre-processing combined with V-ITEX-GC-MS versus direct V-ITEX-GC-MS for urinary volatilome profiling.

Lucie K Tintrop, Doris Zbinden, Marie-Claire Risse, Pascal Fuchsmann.

  Volatile urinary metabolites offer valuable insights into nutritional status and metabolic function. However, the extraction of these metabolites remains analytically challenging due to the complexity and variability of the urine matrix. This study compares and optimizes two extraction techniques, solid-phase extraction followed by vacuum in-tube extraction (SPE+V-ITEX), and direct V-ITEX for the untargeted gas chromatography-mass spectrometry analysis of urinary volatiles. Using design of experiment (DoE) approaches, critical parameters were systematically optimized for both methods. The identified compounds were analyzed with respect to origin, extraction efficiency, and chemical class specificity. Method greenness was assessed using the AGREE and AGREEprep tools. Both SPE+V-ITEX and direct V-ITEX enabled broad metabolite coverage, yielding 1198 detected features, with sufficient repeatability, as 76% of evaluated responses showed RSD values below 20%. SPE+V-ITEX achieved higher total signal intensity for several compounds and compound groups (e.g., alcohols, caprolactam, hetero aromats), and both methods demonstrated sufficient repeatability. Temporal trends in pooled real samples confirmed both methods are applicable for metabolite profiling. Based on AGREE and AGREEprep assessments, V-ITEX emerged as a more sustainable and operationally efficient workflow, aligning closely with green analytical chemistry principles. Desorption flow, trap material, extraction temperature, and time significantly influenced direct V-ITEX performance. Compound-specific trends were observed, but no single method was optimal for all metabolite classes. These findings present direct V-ITEX as a robust, green, and efficient alternative to SPE+V-ITEX for urinary volatilomics and a method particularly well-suited for high-throughput analytical workflows with minimal manual steps.

Keywords:  Metabolomics; Microextraction; Solid-phase extraction; Urine; Vacuum in-tube extraction

DOI:  https://doi.org/10.1007/s00216-026-06469-3
J Chromatogr A. 2026 Mar 24. pii: S0021-9673(26)00261-X. [Epub ahead of print]1776 466931

An integrated derivatization-LC-MS strategy reveals cell-type-specific chiral amino acid profiles.

Yuhan Wang, Jinzheng Liu, Hao Xu, Shuo Yang, Qiang Zhang, Libing Zhang, Yin Xiao.

  Precise analysis of cellular chiral amino acids is critical for elucidating molecular mechanisms in physiology and pathology, yet remains challenging due to complex biological matrices and limited detection sensitivity. To address this, we developed a LC-MS/MS analytical strategy based on dansyl chloride (Dns-Cl) derivatization. This method significantly enhanced separation and ionization efficiency while minimizing matrix interference, enabling the simultaneous trace-level detection and quantification of 19 amino acid enantiomer pairs (with chiral separation and quantification of 16 d/l pairs). The validated method demonstrated excellent analytical figures of merit: low limits of detection (0.007-0.27 nmol), a broad linear range (0.33-33 nmol), high precision (RSD < 6.16 %), and negligible matrix effects (< 6.59 %). Its robust applicability was confirmed through successful analysis of chiral amino acids in fetal bovine serum and multiple cell lines. Metabolite profiling revealed distinct chiral signatures: the contents of d‑serine (d-Ser), d-proline (d-pro), d-methionine (d-Met) were significantly higher in breast cancer MDA-MB-231 cells compared to normal breast MCF-10A cells, suggesting a potential link to malignancy. Furthermore, the d/l enantiomeric ratio of asparagine served as a key discriminant between cancers, with MDA-MB-231 cells enriched in the d-form and HepG2 cells in the l-form. Extracellular chiral amino acid patterns also varied, reflecting cell-type-specific differences in uptake and excretion. Collectively, this work establishes a reliable analytical method and highlights chiral amino acid profiling as a powerful tool for cellular molecular phenotyping, offering novel perspectives for disease mechanism research and biomarker discovery.

Keywords:  Cellular analysis; Chiral amino acid profiles; Dansyl-derivatization; Enantiomeric separation; LC-MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2026.466931
Anal Chem. 2026 Apr 03.

Integrated Single-Cell Mass Spectrometry Imaging and Immunofluorescence Microscopy for Multimodal Characterization of Human Immune Cells.

Sebastian Bessler, Mathis Richter, Jan Schwenzfeier, Sara N Reinartz Groba, Jonel Trebicka, Klaus Dreisewerd, Jens Soltwisch, Oliver Soehnlein.

Understanding how individual cells differ within complex biological systems is essential for revealing mechanisms of health and disease. Multimodal single-cell technologies provide powerful tools for this task, yet single-cell lipid profiling by mass spectrometry imaging often faces technical challenges such as batch effects that obscure biological insights. In this study, we developed an integrated workflow that combines immunofluorescence-based protein measurements with mass spectrometry imaging-based lipid analysis at single-cell resolution. We applied this approach to circulating human neutrophils and implemented a strategy to reduce batch-related variability, enabling more reliable comparison across clinical samples. Using the integrated data set, we identified distinct signatures marking the emergence of pathogenic neutrophil populations in patients with liver cirrhosis. These findings demonstrate the value of combining multimodal single-cell profiling with batch-effect correction to discover cellular phenotypes and highlight the potential of this technology for translational and clinical research.

DOI: https://doi.org/10.1021/acs.analchem.6c00381
ACS Chem Neurosci. 2026 Mar 30.

Spatial Lipidomics Reveals Region-Specific Lipid Remodeling in Scn2a-Deficient Mouse Brain.

Alyssa Moore, Xiaoling Chen, Emerson Hernly, Jingliang Zhang, Yang Yang, Julia Laskin.

  Mass spectrometry imaging (MSI) is a powerful tool for mapping the spatial distribution of biomolecules in biological samples. Among the various MSI techniques, nanospray desorption electrospray ionization (nano-DESI) is ideally suited for quantitative imaging of a wide range of biomolecules in biological tissues due to its capabilities as an ambient, liquid extraction-based technique. In this study, we used nano-DESI MSI to investigate the effects of Scn2a gene deficiency in the mouse brain. Scn2a, which encodes the voltage-gated sodium channel NaV1.2, is critical to neuronal excitability, and its dysfunction is linked to epilepsy and neurodevelopmental disorders such as autism. Despite its importance, the molecular alterations associated with Scn2a dysfunction are still poorly understood. Herein, we present the first comprehensive study of regional lipid and metabolite alterations associated with Scn2a deficiency, achieved by comparing brain tissues from wild-type (WT) and Scn2a homozygous gene-trap (HOM) mice. Nano-DESI MSI experiments were performed on an Orbitrap mass spectrometer in both positive and negative ionization modes, with three biological replicates per group to ensure reproducible detection and broad coverage of biomolecules. Region-of-interest (ROI) analysis revealed multiple species with altered abundance in the HOM mouse brain. Notably, several phosphatidylethanolamine (PE) lipids were observed at higher abundance in different regions of the brain. For example, PE(O-36:5) is more abundant in both the cortex and hippocampus of the HOM brains, while PE(40:4) is more abundant in the hippocampus. Meanwhile, several lipid species, including phosphatidylserine, PS(38:1), were at lower abundance in the cortex. In contrast, abundant structural lipids, including phosphatidylinositol, PI(38:4), and phosphatidylcholine, PC(34:1), showed no significant differences between WT and HOM brains. Our findings offer new insights into the lipid alterations underlying epilepsy and related neurodevelopmental disorders associated with Scn2a deficiency.

Keywords:  epilepsy; lipidomics; mass spectrometry imaging; nano-DESI; neurodevelopmental disorders; spatial neurobiology

DOI:  https://doi.org/10.1021/acschemneuro.6c00199
Methods Mol Biol. 2026 ;3026 11-24

Simultaneous Quantification of Salicylic Acid, Pipecolic Acid, and N-Hydroxypipecolic Acid in Plants Using High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Lingya Yao, Xiu-Fang Xin, Christian Danve M Castroverde.

  Plant immune activation in response to disease-causing pathogens entails proper spatiotemporal regulation of plant hormones and defense-activating metabolites. In Arabidopsis thaliana and numerous plant species, disease resistance to biotrophic and hemibiotrophic pathogens is underpinned by accumulation of these important immune signals, which include salicylic acid (SA), pipecolic acid (Pip), and N-hydroxypipecolic acid (NHP). SA, Pip, and NHP are essential for activating plant defense against various pathogens in local infected tissues, as well as priming distal, uninfected plant tissues in a phenomenon called systemic acquired resistance (SAR). Therefore, accurate quantification of these defense molecules is required to fully investigate the detailed regulation and function of plant immune networks. Here we present a highly sensitive and simple procedure to simultaneously quantify these immune-activating metabolites using state-of-the-art, high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS). A detailed pipeline is presented, encompassing proper plant growth, pathogen infection, metabolite extraction, metabolite quantification, and data analyses. Overall, our current protocol enables accurate measurements of these molecules at low, physiologically relevant concentrations in plant tissues.

Keywords:  Amino acid; HPLC; Mass spectrometry; Metabolite quantification; N-hydroxypipecolic acid; Pipecolic acid; Plant hormone; Plant immunity; Salicylic acid; Systemic acquired resistance

DOI:  https://doi.org/10.1007/978-1-0716-5214-5_2
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Mar 22. pii: S1570-0232(26)00112-1. [Epub ahead of print]1276 125023

Development and validation of an LC-MS/MS method for the analysis of total ascorbic acid capacity in plasma.

Dylan Kelly, Claude P van der Ley, Ron Booij, Ido P Kema, Michel J Vos, Martijn van Faassen.

  A robust and fast LC-MS/MS method for quantifying total ascorbic acid (vitamin C) in plasma was developed and validated. Ascorbic acid is vital for antioxidant defense and enzymatic processes. Deficiency and hypovitaminosis remain prevalent in specific patient groups and can result in overlooked or misattributed clinical symptoms. To preserve ascorbic acid after venapuncture, EDTA blood samples were kept on ice and processed within two hours. Dehydroascorbic acid was reduced to ascorbic acid using tris(2-carboxyethyl)phosphine (TCEP). Ascorbic acid was separated by reversed-phase chromatography and detected by tandem mass spectrometry in negative ion mode. Method validation followed standard protocols. Retrospective analysis of 547 patient samples, analyzed with this method, was performed to assess the incidence of vitamin C deficiency and hypovitaminosis.A TCEP concentration of 15 mM was sufficient. Ascorbic acid eluted at 1.08 min, with a total runtime of 4.6 min. Intra- and inter-assay imprecision (three concentrations) ranged from 1.8 to 2.2% and 3.2-5.7%, respectively. The limit of quantification was 1.1 μmol/L, suitable for detecting deficiency (<11.4 μmol/L). Among the tested patients, 10% were deficient and 20% had hypovitaminosis. Males had ∼11 μmol/L lower vitamin C levels than females, independent of age.This validated LC-MS/MS method enables accurate assessment of total ascorbic acid in clinical settings. With a significant proportion of patients showing suboptimal vitamin C levels, increased awareness is needed to support targeted screening and nutritional interventions.

Keywords:  Ascorbic acid; Chromatography; Hypovitaminosis; LC-MS/MS; Mass spectrometry; Plasma; TCEP; Validation; Vitamin C

DOI:  https://doi.org/10.1016/j.jchromb.2026.125023
Anal Chim Acta. 2026 Jun 01. pii: S0003-2670(26)00268-0. [Epub ahead of print]1401 345318

Meeting the analytical challenges of fatty-acid engineering: a comprehensive method for triacylglycerol identification and quantitation in omega-3 enhanced oilseeds.

Daria Makeeva, Susana Silvestre, Johnathan A Napier, Richard P Haslam, Louise V Michaelson.

   BACKGROUND: The structural elucidation and quantification of complex triacylglycerol (TAG) mixtures remain a major challenge in lipidomics due to extensive isomeric and isobaric diversity arising from differences in fatty-acyl chain length, unsaturation, and positional arrangement. Genetically engineering oilseeds to produce long-chain omega-3 fatty acids further expands this complexity by introducing numerous novel and low-abundance TAG species. A comprehensive method to quantify a wide diversity of TAG is required [67].
RESULTS: To address these analytical demands, we developed a robust workflow using a Q-Exactive Orbitrap platform that integrates untargeted data dependent acquisition (DDA) with targeted parallel reaction monitoring (PRM) for accurate identification and quantification of TAG in genetically modified Camelina sativa engineered to synthesize EPA (20:5) and DHA (22:6). The combined DDA-PRM strategy, supported by retention-time validation, resolved 86% of isobaric TAG pairs and enabled confident assignment of 162 fully fatty-acid resolved TAG species. This is the highest number reported for any plant oil to date, including TAG containing non-native fatty-acids 22:6, 20:5, 22:5, 18:4, and 20:4 which were generated from the engineered enzyme activities. Quantitative accuracy was achieved using a response-factor approach that corrects for ionization efficiency differences, with validation against orthogonal GC-FID analysis [125].
SIGNIFICANCE: The method provides four orders of linear dynamic range, exceeding previous PRM-based lipidomics on Q-TOF instruments. Custom scripts for response-factor calculation and PRM scheduling improved throughput and scalability. This integrated workflow bridges discovery driven lipidomics and high-precision quantitation, offering a versatile and reliable platform for TAG profiling in applications spanning plant metabolic engineering, nutritional quality assessment, and food authenticity [58].

Keywords:  Camelina sativa; Mass spectrometry; Parallel reaction monitoring; Targeted lipidomics; Triacylglycerols

DOI:  https://doi.org/10.1016/j.aca.2026.345318
J Am Soc Mass Spectrom. 2026 Apr 01.

Improved Methods for Recording Accurate Collision Cross Sections Using Cyclic Ion Mobility-Mass Spectrometry.

Devin M Makey, David Langridge, Keith Richardson, Ryan Schroeder, Jakub Ujma, Kevin Giles, Brandon T Ruotolo.

Traveling wave ion mobility (TWIM) is a gas-phase separation technique widely used in structural biology to rapidly probe protein conformational states and in complex mixture analysis to catalyze an array of chemical measurements. Analyte size information can be extracted from TWIM data as collision cross section (CCS) values, which are physicochemical descriptors of the analytes. However, calibration using ions of known CCS is currently required. Recently, an improved TWIM calibration approach was introduced that incorporates blend and radial expressions derived from velocity relaxation and axial confinement effects. This approach offers several benefits over conventional power-law calibrations, including accurate calibration over a wide range of separation conditions and analyte classes using a universal calibrant set. While the blend and radial calibrations were extensively validated using a linear TWIM separator, next-generation cyclic ion mobility (cIM) offers additional benefits, such as scalable resolution using multipass separations and the ability to subject ions to multiple stages of activation and separation (i.e., IMSn). The original work provided a brief application of the blend and radial expressions to cIM calibrations, but these cIM CCS calibrations were applied only to a basic single-pass mode of operation under limited separation conditions. Here, we describe a comprehensive method for accurate cIM calibrations using blend and radial expressions. Correcting ion arrival times for the time spent outside the separator and using conditions that minimize velocity relaxation effects reduced CCS measurement error. We also provide an optimized workflow for calibration using the blend and radial expressions in multipass and IMSn modes of operation.

DOI: https://doi.org/10.1021/jasms.6c00022
J Chromatogr A. 2026 May 24. pii: S0021-9673(26)00248-7. [Epub ahead of print]1775 466918

Assessment of the inaccuracy associated with the quantification of phenolic compounds using UHPLC-DAD and HPLC-MS/MS.

Rosa Zapata, Francisco Artés-Hernández.

  The growing emphasis on health-conscious dietary practices has stimulated extensive research of phenolic and antioxidant compounds, particularly within horticultural food matrices. Their individual quantification, following separation and identification is commonly performed by (ultra-)high-performance liquid chromatography -(U)HPLC- coupled with UV-vis absorption or mass spectrometry. Given the extensive chemical diversity and the economic cost of high-purity standards, the use of equivalent standards to quantify multiple analytes with similar structures is a widespread practice. Despite the evident inaccuracy this approximation may introduce, it is scarcely elucidated in scientific literature. To clarify the magnitude of this error, external standard calibration was performed for 6 phenolic acids and 8 flavonoids using 2 liquid chromatographs, a UHPLC coupled with a diode array detector (DAD) and an HPLC with tandem mass spectrometry (HPLC-MS/MS). When calculations were expressed in terms of molarity, lower discrepancies were obtained by accounting for the molecular weight difference. The UHPLC-DAD analyses exhibited a lower degree of inaccuracy, particularly for flavonoids, no differences were observed when using eriocitrin, rutin, or luteolin to quantify a 16.8 µM eriocitrin solution, with slight variations for all. In contrast, HPLC-MS/MS showed different concentration values across all standards, except between naringenin and diosmin, and luteolin and rutin, which could be considered mutually equivalent. This study provides key insights into the relation between structural diversity and signal using UV-Vis absorption and mass spectrometry representing a starting point for future research focused on the accurate individual quantification of food compounds within their wide diversity.

Keywords:  Absorptivity; Diglycosylated flavanone; Flavonoid aglycone; Hydroxycinnamic acids; Multiple reaction monitoring; Quasi-molecular ion

DOI:  https://doi.org/10.1016/j.chroma.2026.466918
Anal Chem. 2026 Mar 30.

Achieving Single-Cell Resolution via Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) on Different Platforms.

Nathan Colwell, Dan Chen, Deepti Bhusal, Zongkai Peng, Zhibo Yang.

Desorption electrospray ionization (DESI) is a widely used ambient mass spectrometry imaging (MSI) technique valued for its minimal sample preparation and ability to preserve native chemical states. However, achieving single-cell resolution with DESI has been challenging due to relatively low efficiencies of molecular ionization and ion transmission at small spatial scales. Here, we present four distinct implementations that enable single-cell DESI imaging of cultured cells through a combination of optimized experimental parameters and modular hardware integration. In the first platform, a Waters system consisting of a DESI XS source and a Synapt G2-Si Q-TOF mass spectrometer was used with a customized heated ion-transfer capillary and carefully optimized key parameters, including heating temperature, sprayer-to-surface distance, and solvent flow rate, for improved desolvation and ion transmission. In the second platform, a home-built sampling and ionization setup, including a DESI XS sprayer, motorized XYZ-stage, microscope, and ion-source interface, was coupled to a Thermo LTQ Orbitrap XL mass spectrometer. In the third platform, a similar setup containing a DESI XS sprayer was integrated with a Thermo Exploris 240 Orbitrap mass spectrometer. In the fourth platform, a similar setup was coupled to a Thermo Orbitrap Fusion Lumos mass spectrometer. All four platforms allowed MSI studies of metabolites in single cells with heterogeneous populations. Integration with Orbitrap systems provided higher mass resolution and improved spatial resolution, facilitating a demonstration of DESI-based single-cell MSI. Among all four platforms, combining DESI XS source with Exploris 240 resulted in the smallest pixel size (2.7 μm × 10 μm) and largest number of detected molecular features. Together, these results establish a flexible and reproducible framework for adapting DESI across platforms for high-resolution ambient MSI and reveal distinct chemical differences between neighboring cells under native conditions.

DOI: https://doi.org/10.1021/acs.analchem.5c08032
J Mass Spectrom. 2026 Apr;61(4): e70055

Ultra-Performance Liquid Chromatography-Electrospray Ionization-Ion Trap Tandem Mass Spectrometry for Fragmentation of Cyclic Dipeptides: Methodology and Application to the Chemical Profiling of the Fungus Hormonema dematioides.

Min Jae Kim, Xinyun Zhang, Zhao Yang, Xiaofan Fan, Hongjuan Pan, Zhihong Cheng.

  Cyclic dipeptides (CDPs), distinguished by a 2,5-diketopiperazine core, exhibit a broad spectrum of biological activities. The (un)targeted discovery of CDPs in complex matrices is therefore of considerable interest, and mass spectrometry (MS) offers unrivalled advantages: rapid analysis, high sensitivity, minimal sample consumption, and limited pre-treatment requirements. Yet, confident MS-based identification of CDPs has been hampered by the scarcity of authentic reference standards. To address this deficit, we undertook a systematic investigation of 21 CDPs isolated from the fungus Hormonema dematioides using ultra-performance liquid chromatography-electrospray ionization-linear ion trap multistage MS (UPLC-ESI-LIT-MSn) in positive ion mode. Hierarchical MS2 and MS3 experiments afforded reproducible fragmentation pathways that unambiguously delineated the CDP nucleus and the constituent amino acid residues. For the first time, we herein proposed [M + H-17]+ and [M + H-56]+ as co-diagnostic ions for the 2,5-diketopiperazine scaffold, together with the previously reported [M + H-28]+, [M + H-45]+, and [M + H-73]+ ions. Additionally, in contrast to previous reports, the [M + H-28]+ ion consistently appeared as the base peak for the majority of CDPs examined. Amino-acid-specific fragments were found to be strictly dependent on the nature of the incorporated residues. These fragmentation rules were subsequently applied to the untargeted profiling of H. dematioides, enabling the identification or tentative annotation of 30 CDPs through combined UV and MSn (n = 3) data, corroborated wherever possible by authentic standards. Notably, 10 of the CDPs are reported here for the first time from this fungal species.

Keywords:   Hormonema dematioides ; 2,5‐diketopiperazine; LC–MSn; cyclic dipeptides; fragmentation; ion trap

DOI:  https://doi.org/10.1002/jms.70055
Talanta. 2026 Mar 30. pii: S0039-9140(26)00401-7. [Epub ahead of print]306 129745

Analysis of ultra-short-chain and short-chain PFAS in environmental samples: A review.

Toyin Dunsin Saliu, Termeh Teymoorian, Gabriel Munoz, Marie-Claire Grenon, Sébastien Sauvé.

  Ultra-short-chain (USC) and short-chain (SC) per- and polyfluoroalkyl substances (PFAS) are increasingly reported in environmental samples, but their quantitative analysis remains challenging. The measurement process often suffers from poor chromatographic retention, elevated background contamination, and matrix interferences. Environmental monitoring of USC PFAS has also been limited to a few perfluoroalkyl acids, such as trifluoroacetic acid (TFA), perfluoropropanoic acid (PFPrA), and C1-C3 perfluoroalkane sulfonates, while neglecting a plethora of USC precursors originating from aqueous film-forming foams, fluorinated ionic liquids (e.g., bistriflimide), and other applications. In this study, we reviewed findings from about 200 articles reporting USC and SC PFAS, with a specific focus on analytical methods and challenges. Key sample preparation steps reported for effective quantification of USC and SC PFAS included, but were not limited to, the use of PFAS-free labware, proper sample storage, isotope dilution, mild sample acidification before solid-phase extraction (off-line or on-line SPE), and specific cleanup procedures to eliminate matrix interferences. Approximately one-third of the reviewed studies employed reversed-phase liquid chromatography (RPLC), often resulting in poor sensitivity and inadequate retention of TFA. Preferred chromatographic modes for USC PFAS were hydrophilic interaction liquid chromatography (HILIC) and mixed-mode anion exchange liquid chromatography (AEX). Analytical figures of merit, including contamination and filtration artifacts, extraction efficiency, and limits of detection, are critically discussed. A list of ultra-short-chain PFAS relevant to environmental screening is also provided.

Keywords:  Anion-exchange chromatography (AEX); Bistriflimide; Hydrophilic interaction liquid chromatography (HILIC); Short-chain PFAS; Trifluoroacetic acid (TFA); Ultra-short-chain PFAS

DOI:  https://doi.org/10.1016/j.talanta.2026.129745
J Chromatogr A. 2026 May 24. pii: S0021-9673(26)00245-1. [Epub ahead of print]1775 466915

Combining intact and oxidative LC-MS/MS methods to mitigate health risks from Ostreopsis cf. ovata.

Chiara Melchiorre, Martina Carelli, Valeria Tegola, Samuela Capellacci, Silvia Casabianca, Antonella Penna, Carmela Dell'Aversano.

  Ostreopsis cf. ovata is a benthic marine dinoflagellate which produces highly potent neurotoxins named ovatoxins (OVTXs), with OVTX-a being the dominant variant in the Mediterranean strains. OVTXs are associated with respiratory issues via aerosol inhalation and skin/eye irritation from direct seawater contact. Recently, FAO-IOC-IAEA guidelines indicated potential health risk when O. cf. ovata concentrations exceed 3 × 104 cells L⁻¹ in seawater and 5 × 105 cells g-1 in macroalgae. Routine monitoring programs focus only on cell counts and do not measure OVTX-levels, limiting the accuracy of health risk assessment. Herein, we detected and quantified ovatoxins in two of six samples collected along Campania coast by a combination of intact and oxidative sample preparation and Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS). OVTXs concentration in seaweed wash seawater was in the range 1340-1497 ng/mL for Aragonese Castle-L sample and 60-107 ng/mL for Gaiola sample. Matrix effects ranged from -31 % to 2 %, and recovery yields from 41 % to 97 %. After correction for matrix effects and procedural losses, OVTX concentrations were estimated at 13.9 nM (Aragonese Castle-L) and 2.1 nM (Gaiola), values within the cellular toxicity range. However, the abundance of O. cf. ovata cells on macroalgae (6.4 × 10⁴ and 1.6 × 10⁴ cells g⁻¹) remained below the alert threshold. These results highlight the need to integrate toxin quantification into O. cf. ovata monitoring programs. Given the current lack of OVTX reference materials, the use of both intact and oxidative methods combined to LC-MS/MS is a valid strategy for effective risk assessment.

Keywords:  Intact method; LC- MS/MS; Ostreopsis cf. ovata; Ovatoxin – a; Oxidative method

DOI:  https://doi.org/10.1016/j.chroma.2026.466915