bims-metlip 2025-04-27 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–04–27
thirty-one papers selected by
Sofia Costa, Matterworks

Quantitative Analysis of Bimiralisib in Dried Blood Spots of Mouse Blood Using a Validated LC-MS/MS Method: An Application to Pharmacokinetic Study.
Mass spectrometry-based characterisation of the cardiac microtissue metabolome and lipidome.
A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices.
LC-MS and High-Throughput Data Processing Solutions for Lipid Metabolic Tracing Using Biorthogonal Click Chemistry.
Extended Steroid Profiling in Human Serum and Plasma With Simultaneous Quantitative Determination Using One-Point Internal Calibration.
Determination of Thyroid Hormones and 11 Metabolites in the Human Serum Using a Simple Derivatization Strategy and Analysis by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry.
Green Extraction Method: Microwave-Assisted Water Extraction Followed by HILIC-HRMS Analysis to Quantify Hydrophilic Compounds in Plants.
Determination of human urinary cortisol, cortisone and relative phase II metabolites by dilute-and-shoot LC-MS/MS analysis: an application to adrenal disease.
Mass Spectrometry-based Single-Cell Lipidomics: Advancements, Challenges, and the Path Forward.
A rapid method for determination of rosuvastatin in blood plasma with supported liquid extraction.
Liquid Chromatography Combined With Electrospray Ionization Tandem Mass Spectrometry for the Determination and Identification of AZD5462 and Its Metabolites in Rat Plasma.
Exploring the Most Effective Strategy for Purine Metabolite Quantification in Veterinary Medicine Using LC-MS/MS.
Integrating the Preparation of a Tissue Section on Adhesive Tape with an Adsorption Platform Device for Simplified Ambient Mass Spectrometry Imaging Analysis.
Towards Optimizing Neural Network-Based Quantification for NMR Metabolomics.
Highly Sensitive Chemical Ionization Tandem Mass Spectrometry Method for the Identification of Unsaturated Fatty Acids Derivatized by Dimethyl Disulfide.
Identification and quantitative measurement of pyroglutamic acid in 1H NMR spectra of wine.
High-Throughput Screening of Polyfluoroalkyl Substances Using Solid-Phase Microextraction Coupled to Microfluidic Open Interface-Mass Spectrometry.
A parallel reaction monitoring-mass spectrometric method for studying lipid biosynthesis in vitro using 13C16-palmitate as an isotope tracer.
Simultaneous Determination of Multiple Anti-Seizure Medications in Human Plasma Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry with Positive/Negative Ion-Switching Ionization Mode for Therapeutic Drug Monitoring.
The power of trapped ion mobility for isotope tracer experiments.
Protocol to quantitatively assess glycolysis and related carbon metabolic fluxes using stable isotope tracing in Crabtree-positive yeasts.
Met-ID: An Open-Source Software for Comprehensive Annotation of Multiple On-Tissue Chemical Modifications in MALDI-MSI.
Challenges in Spatial Metabolomics and Proteomics for Functional Tissue Unit and Single-Cell Resolution.
An approach of molecular-fingerprint prediction implementing a GAT.
Direct analysis of biotransformations with mass spectrometry-DiBT-MS.
Investigation of polyphenol diversity among lentil species (Lens spp.) using mass spectrometry-based metabolomics guided by photodiode array detection.
A Simple and Sensitive LC-MS/MS Method for Determination of Dapagliflozin and Its Major Metabolite in Human Plasma.
Maximizing Data Coverage through Eight Sequential Mass Spectrometry Images of a Single Tissue Section.
Improving lipidomic coverage for animal matrices by optimizing extraction methods and resuspension solvents based on UHPLC-Q-Exactive Orbitrap MS/MS.
Mass spectral database-based methodologies for the annotation and discovery of natural products.
A multimodal mass spectrometry imaging workflow for ballpoint pen ink analysis and "forgery" detection.

Biomed Chromatogr. 2025 Jun;39(6): e70087

Quantitative Analysis of Bimiralisib in Dried Blood Spots of Mouse Blood Using a Validated LC-MS/MS Method: An Application to Pharmacokinetic Study.

Ashok Zakkula, M Akiful Haque.

  Bimiralisib, a pan-PI3K/mTOR inhibitor, has demonstrated antitumor efficacy in preclinical models. In this study, we present a validated LC-MS/MS method for quantifying bimiralisib from dried blood spots (DBS) in mice. The method was validated in accordance with FDA guidelines. Bimiralisib was extracted from DBS disks using a liquid-liquid extraction technique. Chromatographic separation was achieved on an Atlantis dC18 column (100 × 4.6 mm) using an isocratic mobile phase. The flow rate was set at 0.70 mL/min. Under optimized conditions, the retention times for bimiralisib and the internal standard (IS, Nilotinib) were approximately 1.14 and 1.27 min, respectively, with a total run time of 2.00 min per injection. The monitored MS/MS ion transitions were m/z 412.2 → 141.0 for bimiralisib and 530.4 → 259.0 for the IS. The method employed a broad calibration range (1.00-1434 ng/mL) with a determination coefficient (r2) of 0.996. All validation parameters met the required acceptance criteria, and hematocrit levels had no impact on bimiralisib concentrations in DBS. The validated method was utilized to determine intravenous and oral pharmacokinetic parameters by quantifying bimiralisib in mouse blood, with results correlated to pharmacokinetic data from mice plasma.

Keywords:  DBS; LC‐MS/MS; bimiralisib; method validation; mouse blood; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70087
Metabolomics. 2025 Apr 21. 21(3): 54

Mass spectrometry-based characterisation of the cardiac microtissue metabolome and lipidome.

Tara J Bowen, Andrew R Hall, Andrew D Southam, Ossama Edbali, Ralf J M Weber, Amanda Wilson, Amy Pointon, Mark R Viant.

   INTRODUCTION: The use of large, non-sample specific metabolite reference libraries often results in high proportions of false positive annotations in untargeted metabolomics.
OBJECTIVE: This study aimed to measure and curate a library of polar metabolites and lipids present in cardiac microtissues.
RESULTS: Untargeted ultra-high performance liquid chromatography-coupled mass spectrometry measurements of cardiac microtissue intracellular extracts were annotated by comparison against four spectral databases and a retention time library. The annotations were combined to create a library of 313 polar metabolites and 1004 lipids.
CONCLUSIONS: The curated library will facilitate higher confidence metabolite annotation in mass spectrometry-based untargeted metabolomics of cardiac microtissues.

Keywords:  Cardiac microtissues; Metabolome annotation; Metabolomics; UHPLC-MS

DOI:  https://doi.org/10.1007/s11306-025-02252-0
Sci Rep. 2025 Apr 23. 15(1): 14187

A mixed-mode LC-MS-based method for comprehensive analysis of NAD and related metabolites from biological sample matrices.

Omprakash Nacham, Jordan W Brown, Mohammad Mehdi Maneshi, Victoria Kurschner, Mike Sheehan, Renee Sadowski, Chris Ling, Nari Talaty, Robert Johnson, Andrew M Swensen.

  Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite contributing to cellular energy needs and its decline is associated with age-related disorders. Comprehensive analysis of the NAD+ landscape following NAD+ supplementation therapies would provide a broader understanding of impacts on NAD+ pathway biology. However, the analysis of NAD+ and its metabolites is challenging owing to their polar nature and low retention on reverse phase columns. We have developed and optimized a mixed-mode (reverse-phase/anion-exchange) chromatography-tandem mass spectrometry (LC-MS/MS) method for analysis of NAD+ precursors and their metabolic products from biological sample matrices. Attributes including mobile phase ionic strength and column temperature effects on LC-MS/MS performance were evaluated. Fit-for purpose method qualification was performed with regard to linearity, accuracy, and precision. The method described was developed to be compatible with NAD-Glo assay (bioluminescence-based plate reader assay) conditions for purposes of further validating NAD-Glo and allow for expanded NAD+ pathway profiling in NAD-Glo samples. A strong correlation (R2 = 0.94) was demonstrated between the two assays for tissue NAD+ measurements in mice treated with NAM supplementation. The LC-MS/MS and NAD-Glo data confirmed dose-dependent NAD+ boosting in mice lung and skin tissues after NAM treatment. In addition, LC-MS/MS analysis revealed that the highest dose of NAM (900 mg/kg) significantly increased NR, NMN, ADPR, NAM, and m-NAM levels. Overall, we present an LC-MS/MS based orthogonal platform to confirm NAD-Glo data and show applicability of the method to more broadly evaluate the NAD+ metabolome.

Keywords:  Mixed mode chromatography; NAD-Glo; NADomics; Nicotinamide; Nicotinamide adenine dinucleotide; Targeted mass spectrometry

DOI:  https://doi.org/10.1038/s41598-025-97834-2
Angew Chem Int Ed Engl. 2025 Apr 24. e202501884

LC-MS and High-Throughput Data Processing Solutions for Lipid Metabolic Tracing Using Biorthogonal Click Chemistry.

Palina Nepachalovich, Stefano Bonciarelli, Gabriele Lombardi Bendoula, Jenny Desantis, Michela Eleuteri, Christoph Thiele, Laura Goracci, Maria Fedorova.

  Tracing lipid metabolism in mammalian cells presents a significant technological challenge due to the vast structural diversity of lipids involved in multiple metabolic routes. Biorthogonal approaches based on click chemistry have revolutionized analytical performance in lipid tracing. When adapted for mass spectrometry (MS), it enables highly specific and sensitive analyses of lipid transformations at the lipidome scale. Here, we advance this approach by integrating liquid chromatography (LC) prior to MS detection and developing a software-assisted workflow for high-throughput data processing. LC separation resolved labelled and unmodified lipids, enabling qualitative and quantitative analysis of both lipidome fractions, as well as isomeric lipid species. Using synthetic standards and endogenously produced alkyne lipids, we characterized LC-MS behaviour, including preferential adduct formation and extent of in-source fragmentation. Specific fragmentation rules derived from tandem MS experiments for 23 lipid subclasses, were implemented in Lipostar2 software for high-throughput annotation and quantification of labelled lipids. Applying this platform, we traced metabolic pathways of palmitic and oleic acid alkynes, revealing distinct lipid incorporation patterns and metabolic bottlenecks. Altogether, here we provide integrated analytical and bioinformatics platform for high-throughput tracing of lipid metabolism using LC-MS workflow.

Keywords:  click chemistry * LC-MS * Lipostar2 * sphingolipids * tracing lipid metabolism

DOI:  https://doi.org/10.1002/anie.202501884
J Sep Sci. 2025 Apr;48(4): e70147

Extended Steroid Profiling in Human Serum and Plasma With Simultaneous Quantitative Determination Using One-Point Internal Calibration.

Mathieu Galmiche, Oriane Strassel, Marie-Anaïs Monat, Isabel Meister, Julien Boccard, Serge Rudaz.

  Steroids are a major set of endogenous bioactive compounds. Although increasingly popular, their analysis in biofluids by LC-MS is associated with enduring challenges, such as their low endogenous concentrations or the coexistence of numerous isobaric compounds. Their natural presence in biological matrices complicates their absolute quantification in blood, as the obtention of a blank matrix to establish an external calibration curve is impossible. This protocol describes a strategy for developing an LC-MS/MS method for the extended profiling of steroids in serum and plasma, including as much as 171 target compounds, with the additional absolute quantification of four main steroids (cortisol, testosterone, progesterone, and androstenedione). The proposed sample preparation involves protein precipitation in organic solvents and subsequent filtration of the sample on HLB cartridge. The LC method is developed to resolve most isobaric species thanks to a biphenyl stationary phase. MS detection is performed in multiple reaction monitoring mode with post-column addition of ammonium fluoride to enhance sensitivity. A one-point internal calibration strategy is presented for the absolute quantification of endogenous steroids. The application of this method to the NIST Plasma Reference Material (SRM 1950) led to the identification of 69 distinct endogenous steroids, making it the most comprehensive profiling of these compounds in this reference matrix to date. The quantitative performance of the method is assessed with two certified materials and shows satisfactory precision and trueness.

Keywords:  absolute quantification; endogenous compounds; liquid chromatography; mass spectrometry; steroids

DOI:  https://doi.org/10.1002/jssc.70147
Anal Chem. 2025 Apr 23.

Determination of Thyroid Hormones and 11 Metabolites in the Human Serum Using a Simple Derivatization Strategy and Analysis by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry.

Jiří Kohoutek, Juan I Sánchez-Avila, Marie Smutná, Petr Janků, Jana Klánová, Klára Hilscherová.

Many analytical methods for thyroid hormone (TH) determination lack sensitivity and/or specificity. The thyroid hormone metabolites (THMs) are usually not measured at all. This study describes the development of sensitive high-throughput analytical methods for determining the total concentration and free fraction of TH and THM in the human serum. For the analysis of the TOTAL fraction, we employed protein precipitation and anionic exchanger solid-phase extraction. For the FREE fraction, ultrafiltration and salt-out liquid partitioning were used. Derivatization using dansyl chloride was employed to enhance the sensitivity of HPLC-ESI-MS/MS analysis. Both protocols were validated according to the European Analytical Guidelines (2002/657/EC). We obtained very good recoveries (73-115%) and precision. Interday coefficients of variation (CVs) for most of the analytes ranged from 1.2 to 16.4%. The sensitivity was excellent with detection limits in the sub ppt range for the majority of TH and THM. A significant enhancement in sensitivity (>10 fold) was achieved through derivatization. The applicability was proved on a set of samples from pregnant women enrolled in the CELSPAC cohort (n = 120). Our TH reference ranges are in good agreement with those reported in the literature. The methods also allowed us to quantify the levels of 11 THM, including some previously undetected THM in total and free fractions, and proved to be suitable for high-throughput routine TH and THM analyses. Our approach offers an important advancement in thyroid hormone analysis. To the best of our knowledge, it is for the first time that data for T1A and T2A as well as for free THM levels in the human serum are published in the literature. Moreover, our study also brings the first information about the levels of most of the THM in pregnant women.

DOI: https://doi.org/10.1021/acs.analchem.5c00714
Metabolites. 2025 Mar 25. pii: 223. [Epub ahead of print]15(4):

Green Extraction Method: Microwave-Assisted Water Extraction Followed by HILIC-HRMS Analysis to Quantify Hydrophilic Compounds in Plants.

Alexandra Louis, Jean François Chich, Hadrien Chepca, Isabelle Schmitz, Philippe Hugueney, Alessandra Maia-Grondard.

  Background: Hydrophilic compounds, such as amino acids, organic acids and sugars, among others, are present in large amounts in plant cells. The analysis and quantification of these major hydrophilic compounds are particularly relevant in plant science because they have a considerable impact on the quality of plant-derived products and on plant-pathogen relationships. Our objective was to develop and validate a complete analysis workflow combining a water-based extraction procedure with a fast separation using hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry (HILIC-HRMS) for quantitative analysis of hydrophilic compounds in plant tissues. Results: Water-based microwave-assisted extraction (MAE) methods for hydrophilic compounds were compared using HILIC-HRMS. The newly developed method involved 20 s MAE time followed by a 10 min HILIC-HRMS analysis. This bioanalytical method was validated for 24 polar metabolites, including amino acids, organic acids, and sugars, to ensure the reliability of analytical results: selectivity, limits of detection and quantification, calibration range and precision. Depending on the compounds, quantification limit was as low as 0.10 µM up to 4.50 µM. Between-run RSDs evaluated on Vitis vinifera and Arabidopsis samples were all below 20% except for three compounds. Conclusions: A water-based MAE method, coupled with HILIC-HRMS, was developed for the absolute quantification of free amino acids, organic acids, and sugars in plant tissues. Its effectiveness was demonstrated in both lignified plants, such as Vitis vinifera, and non-lignified plants, such as Arabidopsis. This method is suitable for medium- to high-throughput analysis of key polar metabolites from small amounts of plant material.

Keywords:  amino acids; high-resolution mass spectrometry; hydrophilic interaction liquid chromatography (HILIC); microwave-assisted extraction (MAE); organic acids; primary metabolism

DOI:  https://doi.org/10.3390/metabo15040223
Clin Chim Acta. 2025 Apr 20. pii: S0009-8981(25)00203-7. [Epub ahead of print]574 120324

Determination of human urinary cortisol, cortisone and relative phase II metabolites by dilute-and-shoot LC-MS/MS analysis: an application to adrenal disease.

Laura Leoni, Mirko Parasiliti Caprino, Giulia Montesano, Chiara Lopez, Martina Bollati, Fabio Settanni, Ezio Ghigo, Giulio Mengozzi, Federico Ponzetto.

   BACKGROUND: Cortisol and its primary metabolite, cortisone, play key roles in managing stress responses and maintaining balance via the hypothalamic-pituitary-adrenal axis. Traditional immunoassays, often hindered by cross-reactivity, can yield inaccurate results. This study introduces a novel dilute-and-shoot using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the rapid and accurate analysis of urinary free cortisol, cortisone, and their intact phase II metabolites.
METHODS: A new LC-MS/MS method was developed to quantify five analytes, including intact glucuro- and sulfo-conjugated cortisol and cortisone metabolites. The method underwent validation assessments in accordance with ISO 17025 standards for quantitative methods. Its performance was compared with a commercial LC-MS/MS assay used in clinical laboratories, analyzing free cortisol and cortisone in 79 24-hour urine samples. Additionally, the novel method was applied to authentic urine samples from 168 adrenal disease patients.
RESULTS: The validation protocol assessed method's selectivity, matrix effects, quantitative performance and carry-over. The comparison showed excellent correlation with the commercial LC-MS/MS method for urinary free cortisol and cortisone measurement. Pathological sample analysis indicated that monitoring intact phase II metabolites, particularly sulfoconjugates, could aid in differentiating adrenal diseases.
CONCLUSION: The developed and validated dilute-and-shoot LC-MS/MS method demonstrated satisfactory performance, offering a faster, cost-effective solution for adrenal hormone analysis in clinical settings. This research also provided insights into adrenal function by examining the urinary excretion of target analytes in a pathological cohort, enhancing diagnostic precision for related disorders.

Keywords:  Adrenal; Conjugated; Dilute-and-shoot; LC-MS/MS; Steroids; Urine

DOI:  https://doi.org/10.1016/j.cca.2025.120324
Trends Analyt Chem. 2023 Dec;pii: 117350. [Epub ahead of print]169

Mass Spectrometry-based Single-Cell Lipidomics: Advancements, Challenges, and the Path Forward.

Caitlin E Randolph, Palak Manchanda, Harshit Arora, Sanjay Iyer, Pooja Saklani, Connor Beveridge, Gaurav Chopra.

  In the past decade, lipidomics, now recognized as standalone subdiscipline of metabolomics, has gained considerable attention. Due to its sensitivity and unparalleled versatility, mass spectrometry (MS) has emerged as the tool of choice for lipid identification and detection. Traditional MS-based lipidomics are performed on bulk cell samples. While informative, these bulk-scale cellular lipidome measurements mask cellular heterogeneity across seemingly homogeneous populations of cells. Unfortunately, single cell lipidomics methodology and analyses are considerably behind genomics, transcriptomics, and proteomics. Therefore, the cell-to-cell heterogeneity and related function remains largely unexplored for lipidomics. Herein, we review recent advances in MS-based single cell lipidomics. We also explore the root causes for the slow development of single-cell lipidomics techniques. We aim to provide insights on the pivotal knowledge gaps that have been neglected, prohibiting the propulsion of the single-cell lipidomics field forward, while also providing our perspective towards future methodologies that can pave a path forward.

Keywords:  Matrix-Assisted Laser Desorption/Ionization; ambient ionization; bioanalysis; cellular; cellular heterogeneity; data analysis; electrospray ionization; lipidomics; machine learning; mass spectrometry; mass spectrometry imaging; metabolomics; omics technologies; single-cell analysis; subcellular analysis; tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.trac.2023.117350
J Mass Spectrom Adv Clin Lab. 2025 Apr;36 29-36

A rapid method for determination of rosuvastatin in blood plasma with supported liquid extraction.

Tjaša Dermota, Mojca Božič Mijovski, Jurij Trontelj.

   Introduction: Accurate measurement of rosuvastatin in plasma is critical for effective patient management and treatment monitoring following myocardial infarction (MI). Expensive solid-phase extraction (SPE) and time-consuming liquid-liquid extraction (LLE) have been established for quantifying rosuvastatin. Supported liquid extraction (SLE) could offer a rapid, cost-effective alternative.
Objectives: This study aimed to develop and validate a rapid, cost-effective, accurate, and precise method for quantifying rosuvastatin in high-dose plasma samples from patients following MI.
Methods: Rosuvastatin was extracted from EDTA plasma using SLE and quantified with LC-MS/MS with positive electrospray ionization. The method was validated according to ICH M10 guidelines, focusing on selectivity, matrix effect, accuracy, precision, linearity, and carryover. Rosuvastatin-D6 was used as an internal standard. Additionally, thirty plasma samples from patients on high-dose rosuvastatin therapy (20 or 40 mg/day) following MI were analyzed by both LLE and SLE methods and compared.
Results: The method was successfully validated, demonstrating linearity across a range of 0.1 ng/mL to 50 ng/mL. Compared to the LLE method, SLE achieved superior extraction recovery (96.3 % vs. 60 %) and precision (RSD: 11.9 % vs. 13.6 %) at 0.3 ng/mL rosuvastatin, with a lower absolute matrix effect (12.7 % vs. -36.7 %). Accuracy was comparable (109.3 % vs. 92.8 %). Although SLE involves higher initial costs, it significantly enhances throughput, reduces solvent usage, and minimizes contamination and equipment wear.
Conclusion: This study validates SLE as a superior method for quantifying rosuvastatin in plasma, outperforming LLE in recovery, reproducibility, and automation. SLE offers greater accuracy and reliability, making it ideal for high-throughput applications.

Keywords:  LC-MS/MS; LLE; Rosuvastatin; SLE; Supported Liquid Extraction

DOI:  https://doi.org/10.1016/j.jmsacl.2025.04.003
Biomed Chromatogr. 2025 Jun;39(6): e70095

Liquid Chromatography Combined With Electrospray Ionization Tandem Mass Spectrometry for the Determination and Identification of AZD5462 and Its Metabolites in Rat Plasma.

Congyao You, Yan Zhang, Dece Kong, Tieyi Yang.

  AZD5462, a human RXFP1 agonist, which is undergoing clinical development for the treatment of heart failure. The aim of this study was to develop an ultra-high-performance liquid chromatography-tandem mass spectrometric method for the determination of AZD5462 in rat plasma. After precipitated with acetonitrile, the sample was analyzed on a BEH C18 column using 0.1% formic acid and acetonitrile as mobile phase with a gradient elution at 40°C within 2 min. The assay showed excellent linearity in the range of 0.1-1000 ng/mL with the correlation coefficient more than 0.995. The precision, accuracy, matrix effect, recovery, and stability met all requirements for the quantitation in plasma samples. The validated method has been further applied to the pharmacokinetic study of AZD5462 in rats. In addition, the metabolism of AZD5462 in rat was investigated by a liquid chromatography-high resolution mass spectrometry. In rat liver microsomes, four metabolites were identified based on their accurate mass and fragment ions. In rat plasma, one glucuronide conjugate was identified. The metabolic pathways of AZD5462 include oxygenation and glucuronidation. This study is the first report on the pharmacokinetics and metabolism of AZD5462, which would provide insights into the effectiveness and toxicity of this drug candidate.

Keywords:  AZD5462; RXFP1 agonist; bioavailability; metabolism; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70095
Vet Sci. 2025 Mar 03. pii: 230. [Epub ahead of print]12(3):

Exploring the Most Effective Strategy for Purine Metabolite Quantification in Veterinary Medicine Using LC-MS/MS.

Anisa Bardhi, Francesco Dondi, Andrea Barbarossa.

  Measuring purine metabolites in the serum and urine may help in the early diagnosis of urolith formation, the correction of allopurinol therapeutic dosages, and the evaluation of diet-related alterations. To properly monitor these endogenous compounds and assess their physiological concentration ranges in biological fluids, highly specific and accurate analytical approaches are required. Colorimetric assays are generally used for this purpose, although their cross-reactivity could lead to incorrect determinations. Given the importance of selectivity in detecting endogenous compounds, this study explored the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to enhance uric acid and allantoin measurement in both serum and urine from dogs, as well as urine from bovines. The most effective analytical strategy was identified and successfully applied to an initial batch of samples collected from healthy dogs and bovines. In conclusion, mass spectrometry proved to be a powerful tool for this challenging task, further demonstrating its superior performance in improving clinical laboratory diagnostics, including its valuable applications in veterinary medicine.

Keywords:  allantoin; bovine; clinical pathology; dog; nutrition; serum; standard addition; uric acid; urine

DOI:  https://doi.org/10.3390/vetsci12030230
Anal Chem. 2025 Apr 25.

Integrating the Preparation of a Tissue Section on Adhesive Tape with an Adsorption Platform Device for Simplified Ambient Mass Spectrometry Imaging Analysis.

Yingfeng Xue, Jinyi Li, Zhehui Zhao, Jiaheng Li, Xin Li, Ruiping Zhang, Ling Ren, Lulu Wang, Wenxuan Zhang, Zhigang Luo, Zeper Abliz.

As a visualization technology for the in situ characterization of surface material molecules, mass spectrometry imaging (MSI) analysis is being increasingly used in various fields, especially in the biomedical field. However, the preparation of biological tissue section samples for MSI analysis remains time-consuming and labor-intensive, and sample loss or damage occurs frequently. The inability to stably and consecutively obtain suitable section samples and perform concise and efficient imaging analysis limits the analysis throughput. Herein, a preparation method is proposed. It enables consecutive sectioning, batch preservation, and dry processing through the use of ordinary adhesive tape, enhancing the adhesion between section and tape and rapid freeze-drying. Furthermore, based on the air flow assisted desorption electrospray ionization (AFADESI) MSI system, a vacuum adsorption platform is introduced, which simplifies the process of MSI analysis. Moreover, compared with general tape-based MSI methods, the signal intensity of 73%-85% of the annotated ions is improved for positive ion mode. The signal-to-noise (S/N) ratios of characteristic ions in the corresponding regions in the images of the tissue section samples increase by an average of more than two times, and a clearer organ outline can be seen in the images. By integrating the sample preparation method with the adsorption platform, high-throughput imaging of serial whole-body or scattered organ tissue sections can be conducted more easily and concise and efficient MSI analysis can be performed, which will provide a new strategy to meet rapidly growing MSI research demands.

DOI: https://doi.org/10.1021/acs.analchem.5c01648
Metabolites. 2025 Apr 04. pii: 249. [Epub ahead of print]15(4):

Towards Optimizing Neural Network-Based Quantification for NMR Metabolomics.

Hayden Johnson, Aaryani Tipirneni-Sajja.

  Background: Quantification of metabolites from nuclear magnetic resonance (NMR) spectra in an accurate, high-throughput manner requires effective data processing tools. Neural networks are relatively underexplored in quantitative NMR metabolomics despite impressive speed and throughput compared to more conventional peak-fitting metabolomics software. Methods: This work investigates practices for dataset and model development in the task of metabolite quantification directly from simulated NMR spectra for three neural network models: the multi-layered perceptron, the convolutional neural network, and the transformer. Model architectures, training parameters, and training datasets are optimized before comparing each model on simulated 400-MHz 1H-NMR spectra of complex mixtures with 8, 44, or 86 metabolites to quantify in spectra ranging from simple to highly complex and overlapping peaks. The optimized models were further validated on spectra at 100- and 800-MHz. Results: The transformer was the most effective network for NMR metabolite quantification, especially as the number of metabolites per spectra increased or target concentrations were low or had a large dynamic range. Further, the transformer was able to accurately quantify metabolites in simulated spectra from 100-MHz up to 800-MHz. Conclusions: The methods developed in this work reveal that transformers have the potential to accurately perform fully automated metabolite quantification in real-time and, with further development with experimental data, could be the basis for automated quantitative NMR metabolomics software.

Keywords:  NMR spectroscopy; convolutional neural network; low-field NMR; multi-layered perceptron; transformer

DOI:  https://doi.org/10.3390/metabo15040249
J Am Soc Mass Spectrom. 2025 Apr 21.

Highly Sensitive Chemical Ionization Tandem Mass Spectrometry Method for the Identification of Unsaturated Fatty Acids Derivatized by Dimethyl Disulfide.

Tingxiang Yang, Lerong Qi, Hao Yang, Yihan Xia, Zhen Wang, Dong Hao Wang.

  Derivatization of unsaturated fatty acids with dimethyl disulfide (DMDS) and analysis by electron ionization mass spectrometry (EIMS) represent a convenient offline method for the identification of double bond positions. However, the presence of overlapping mass spectra from multiple compounds poses significant challenges for spectral interpretation and library matching, leading to ambiguous molecular information and low sensitivity. To overcome the issue, we developed a novel chemical ionization (CI) tandem mass spectrometry method involving the pre-derivatization with DMDS and collisional activation of [M+47]+ ions generated in the chemical ion source. The method provides better specificity to the analysis of targeted fatty acids and does not require any customized devices. Further, a multiple reaction monitoring (MRM) version of the method was designed by screening all the diagnostic ions of possible double bond positional isomers, which significantly boosts the sensitivity. Compared to the traditional EIMS method, the new method exhibits a lower limit of detection (LLOD) that is one-tenth or lower. Employing the new method, unusual isomer 18:2(5Z,8Z) was co-analyzed with 18:2(9Z,12Z), and a novel 20:2(7Z,10Z) was characterized in human sebum. Additionally, 16:2(9Z,12Z), an odd-chain omega-3 polyunsaturated fatty acid (21:5n-3) and polymethylene-interrupted isomers, i.e. 22:2(7Z,13Z) and 22:2(7Z,15Z) were identified in seafood and related products. Our method can be readily applied to any GC instrument equipped with tandem MS and is expected to facilitate the discovery and identification of unknown fatty acids from food, clinical, and environmental sources.

Keywords:  Carbon−carbon double bond; Chemical ionization; Dimethyl disulfide; Mass spectrometry; Unsaturated fatty acid

DOI:  https://doi.org/10.1021/jasms.5c00033
Food Res Int. 2025 May;pii: S0963-9969(25)00584-8. [Epub ahead of print]209 116247

Identification and quantitative measurement of pyroglutamic acid in 1H NMR spectra of wine.

Flynn T Watson, Mathias Nilsson, Markus Herderich, Allan Torres, William S Price, Gareth A Morris.

  Proton NMR is one of the key analytical technologies in the field of metabolomics, as it allows one to combine untargeted, targeted, and quantitative metabolite measurements. One of NMR's greatest strengths is the ability to unambiguously identify compounds when present at mg/L concentrations, without the use of expensive or hard-to-source reference compounds. Furthermore, identification can be performed non-destructively on complex samples without the need for further sample preparation and isolation. Here, we describe a series of NMR experiments and data processing techniques to unambiguously identify the metabolite pyroglutamic acid (pGlu) in wine samples, without prior enrichment or separation from matrix compounds and other metabolites typically present in wine. Subsequently, the concentration of pGlu in 100 Australian wines was determined using standard NMR protocols. Statistical analysis demonstrated that occurrence of pGlu is associated with glutamic acid, is linked to vintage conditions and accumulated heat over the growing season, and is negatively associated with rainfall during the growing season. Overall, the results establish the presence and typical concentrations of the amino acid metabolite pGlu in Australian wine.

Keywords:  GEMSTONE; Metabolomics; NMR; Pure shift; Pyroglutamic acid; STOCSY; Ultra-selective; Wine

DOI:  https://doi.org/10.1016/j.foodres.2025.116247
Environ Sci Technol. 2025 Apr 24.

High-Throughput Screening of Polyfluoroalkyl Substances Using Solid-Phase Microextraction Coupled to Microfluidic Open Interface-Mass Spectrometry.

Wei Zhou, Malvika Dutt, Qizhen Lan, Achille Cappiello, Janusz Pawliszyn.

  Efficient and sustainable methods for large-scale PFAS monitoring are critical for addressing environmental and public health challenges. This work presents a high-throughput sample preparation system capable of processing up to 48 samples simultaneously using solid-phase microextraction (SPME) and was directly coupled with mass spectrometry (MS) via an automated microfluidic open interface (MOI), bypassing the need for chromatographic separation. The SPME-MOI-MS approach achieves sensitive detection of 18 PFAS in drinking water, with limits of detection (LODs) between 1 and 10 pg/mL, using just 1.5 mL of sample and an average analysis time of 2.8 min per sample. The SPME blades, employed to enhance sensitivity in place of standard SPME fibers, incorporate a matrix-compatible coating material that enables effective PFAS screening in water as well as complex matrices including blood, beer, and beef. In addition, significantly low recovery and reproducibility of nonpolar PFAS in water analysis have been found and studied, indicating that using a glass container and adding a small percentage of acetonitrile can address this issue.

Keywords:  PFAS; high-throughput; microfluidic open interface; rapid screening; solid-phase microextraction; water analysis

DOI:  https://doi.org/10.1021/acs.est.5c00886
Anal Chim Acta. 2025 Jun 08. pii: S0003-2670(25)00397-6. [Epub ahead of print]1354 344003

A parallel reaction monitoring-mass spectrometric method for studying lipid biosynthesis in vitro using 13C16-palmitate as an isotope tracer.

Kyeong-Seog Kim, Young Gyun Ko, Woo Seok Yang, Hye Young Kim, Joo-Youn Cho.

   BACKGROUND: Palmitate, which is the end product of fatty acid synthase, is the key fatty acid for understanding of lipid biosynthetic process in mammalian cells. Mass spectrometry (MS) methodology using 13C-palmitate can trace the lipid biosynthesis such as glycerolipids, glycerophospholipids, and sphingolipids. However, due to the interferences of natural heavy isotopes, accurate measurement of 13C-labeled lipid species has been limited. Here we describe a high-throughput isotope tracing experiment to assess lipid biosynthesis using parallel reaction monitoring-MS (PRM-MS) with 13C16-palmitate as an isotope tracer.
RESULTS: The developed method can trace 14 13C16-labeled lipid classes without disturbance from the heavy isotope patterns of natural lipids. Lipid class-based separation was achieved through hydrophilic interaction liquid chromatography (HILIC) which allows facile identification of lipid, and PRM-MS was performed for accurate detection of the 13C16-labeled lipids. A fibroblast (NIH/3T3) cell line was used as an in vitro model, and the NIH/3T3 cells were treated with bovine serum albumin (BSA)-bound 13C16-palmitate. The isotopic disturbance from natural lipid was eliminated using 13C16-palmitate, rather than 13C1-palmitate, as an isotope tracer. After 24 h of incubation with 0.1 mmol/L of BSA-bound 13C16-palmitate in the fibroblasts, NIH/3T3 cells synthesized the 127 13C16-labeled lipid species of glycerolipids, glycerophospholipids, and sphingolipids. Finally, in the NIH/3T3 cells incubated for 1, 6, and 24 h after the treatment of the isotope tracer exhibited an increased profile of 13C16-labeled lipidome, depending on duration of incubation.
SIGNIFICANCE: The HILIC/PRM-MS method using 13C16-palmitate as an isotope tracer enables identification of 13C16-labeled lipid species by annotating 13C16-labeled position, including the 13C16-fatty acyl chain and 13C16-sphingolipid headgroup, without interference of natural heavy isotope patterns. This lipidomic flux analysis using PRM approach is expected to provide insights into assessment of isotope-labeled lipids.

Keywords:  HILIC-MS/MS; Isotope tracing; Lipid biosynthesis; Lipidomics; Parallel reaction monitoring

DOI:  https://doi.org/10.1016/j.aca.2025.344003
J Sep Sci. 2025 Apr;48(4): e70151

Simultaneous Determination of Multiple Anti-Seizure Medications in Human Plasma Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry with Positive/Negative Ion-Switching Ionization Mode for Therapeutic Drug Monitoring.

Yu-Fei Zhang, Zheng Yu, Zhao Zhao, Le-Qing Xu, Xun-Yi Wu, Chun-Lai Ma.

  Monotherapy or combination therapy with anti-seizure medications (ASMs) remains the cornerstone of epilepsy treatment. Therapeutic drug monitoring is important for individualized ASM treatment and for optimizing outcomes in patients with epilepsy. Therefore, the simultaneous and accurate quantification of ASMs is necessary and helpful for clinical use. In this study, a new method was developed to simultaneously quantify carbamazepine, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, perampanel, sodium valproate, topiramate, and an active metabolite of oxcarbazepine, 10, 11-dihydro-10-hydroxycarbamazepine, in human plasma by high-performance liquid chromatography-tandem mass spectrometry using the positive/negative ion-switching ionization mode. The method was assessed for accuracy, precision, linearity, recovery, and matrix effects according to the criteria of the U.S. Food and Drug Administration Bioanalytical Method Validation Guidelines for Industry for Bioanalytical Methods. The lower limit of quantification was determined to be 0.001-25 µg/mL for each analyte. The intra- and inter-day precision values had <14.6% coefficients of variation, and the accuracy ranged from 85.3% to 113% at the three levels of quality control. In addition, this method was successfully applied to therapeutic drug monitoring of ASMs in patients with epilepsy. This method allows for simple preparation, accurate quantification, and the determination of different ASMs without requiring analytical method switching, making it particularly suitable for therapeutic drug monitoring in busy clinical and hospital laboratory settings.

Keywords:  HPLC‐MS/MS; anti‐seizure medications; plasma; simultaneous quantification; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/jssc.70151
Anal Chim Acta. 2025 Jun 15. pii: S0003-2670(25)00399-X. [Epub ahead of print]1355 344005

The power of trapped ion mobility for isotope tracer experiments.

Karin Preindl, Chuqiao Chen, Supriya Murthy, Florian Gruber, Christian Freystätter, Thomas Weichhart, Thomas Stimpfl, Birgit Reiter, Arvand Haschemi, Gunda Koellensperger.

   BACKGROUND: Isotope tracing experiments in cellular metabolomics are challenged by the multiple isomers and in-source fragments, which need to be considered to obtain unbiased isotopologue ratio measurements. Thus, both, selectivity and sensitivity are key requirements for customized workflows. Trapped ion mobility spectrometry (TIMS) introduces an additional separation dimension to mass spectrometry, separating otherwise co-eluting isomers by measuring the ion mobility of a molecule. This study shows for the first time, the potential of this MS platform for accurate isotopologue assessment as showcased in isotope tracer experiments using mammalian cells.
RESULTS: The validation exercise focused on spectral accuracy, precision, and metabolite detection capabilities and comprised independent measurements on an orbitrap-based platform. Hydrophilic interaction chromatography, in combination with TIMS-TOF-MS delivered excellent results, with a minimum trueness bias and excellent precision (CV%) between 0.3 % and 6.4 %. The ion mobility separation allowed for differentiation of the otherwise co-eluting isomers fructose-6-phosphate (F6P) and glucose-1-phosphate (G1P). Overall, isotopologue distributions were in good agreement upon crossvalidation with the orbitrap platform. Finally, a proof-of-concept tracer study addressed the activity of the glycolysis and the pentose phosphate pathway (PPP) in resting and endotoxin activated macrophages. We confirmed an activation of glycolysis and PPP in LPS activated macrophages, but found a potentially reduced relative contribution of glucose-6-phosphate (G6P) to increased F6P pools. Our findings imply that TIMS is a powerful technology for the reliable measurements of isotope distribution analysis in metabolic tracing experiments.
SIGNIFICANCE: By implementation of ion mobility, it is now possible to generate distinct isotopologue patterns for G1P and F6P in isotope tracer experiments. F6P plays a crucial role in glycolysis and PPP, highlighting the importance of precise analytical measurements. This is particularly true for metabolic studies in immunology and cancer research.

Keywords:  Isotope tracer; Macrophages; TIMS; Trapped ion mobility

DOI:  https://doi.org/10.1016/j.aca.2025.344005
STAR Protoc. 2025 Apr 22. pii: S2666-1667(25)00192-3. [Epub ahead of print]6(2): 103786

Protocol to quantitatively assess glycolysis and related carbon metabolic fluxes using stable isotope tracing in Crabtree-positive yeasts.

Shreyas Niphadkar, Sreesa Sreedharan, Vineeth Vengayil, Sunil Laxman.

  Crabtree-positive yeasts rapidly consume glucose via glycolysis, making it difficult to experimentally estimate their actual glycolytic rate or flux. We present a stable isotope labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protocol to quantitatively estimate glycolytic and related carbon metabolic fluxes using Saccharomyces cerevisiae. This approach defines time windows to capture glucose metabolic intermediate production before label saturation, enabling a comparison of glycolytic flux changes across different cells. This protocol provides a reliable, quantitative approach to study dynamic metabolic fluxes in these cells. For complete details on the use and execution of this protocol, please refer to Vengayil et al., 2024.1.

Keywords:  metabolism; metabolomics; model organisms; systems biology

DOI:  https://doi.org/10.1016/j.xpro.2025.103786
Anal Chem. 2025 Apr 20.

Met-ID: An Open-Source Software for Comprehensive Annotation of Multiple On-Tissue Chemical Modifications in MALDI-MSI.

Patrik Bjärterot, Anna Nilsson, Reza Shariatgorji, Theodosia Vallianatou, Ibrahim Kaya, Per Svenningsson, Lukas Käll, Per E Andrén.

Here, we introduce Met-ID, a graphical user interface software designed to efficiently identify metabolites from MALDI-MSI data sets. Met-ID enables annotation of m/z features from any type of MALDI-MSI experiment, involving either derivatizing or conventional matrices. It utilizes structural information for derivatizing matrices to generate a subset of targets that contain only functional groups specific to the derivatization agent. The software is able to identify multiple derivatization sites on the same molecule, facilitating identification of the derivatized compound. This ability is exemplified by FMP-10, a reactive matrix that assists the covalent charge-tagging of molecules containing phenolic hydroxyl and/or primary or secondary amine groups. Met-ID also permits users to recalibrate data with known m/z ratios, boosting confidence in mass match results. Furthermore, Met-ID includes a database featuring MS2 spectra of numerous chemical standards, consisting of neurotransmitters and metabolites derivatized with FMP-10, alongside peaks for FMP-10 itself, all accessible directly through the software. The MS2 spectral database supports user-uploaded spectra and enables comparison of these spectra with user-provided tissue MS2 spectra for similarity assessment. Although initially installed with basic data, Met-ID is designed to be customizable, encouraging users to tailor the software to their specific needs. While several MSI-oriented software solutions exist, Met-ID combines both MS1 and MS2 functionalities. Developed in alignment with the FAIR Guiding Principles for scientific software, Met-ID is freely available as an open-source tool on GitHub, ensuring wide accessibility and collaboration.

DOI: https://doi.org/10.1021/acs.analchem.5c00633
Semin Nephrol. 2025 Apr 21. pii: S0270-9295(25)00020-8. [Epub ahead of print] 151583

Challenges in Spatial Metabolomics and Proteomics for Functional Tissue Unit and Single-Cell Resolution.

Kevin J Zemaitis, Ljiliana Paša-Tolić.

  In the last decade, advanced developments of mass spectrometry-based assays have made spatial measurements of hundreds of metabolites and thousands of proteins not only possible, but routine. The information obtained from such mass spectrometry imaging experiments traces metabolic events and helps decipher feedback loops across anatomical regions, connecting genetic and metabolic networks that define phenotypes. Herein we overview developments in the field over the past decade, highlighting several case studies demonstrating direct measurement of metabolites, proteins, and proteoforms from thinly sliced tissues at the level of functional tissue units, approaching single-cell levels. Much of this work is feasible due to multidisciplinary team science, and we offer brief perspectives on paths forward and the challenges that persist with adoption and application of these spatial omics techniques at the single-cell level on mammalian kidneys. Data analysis and reanalysis still pose issues that plague spatial omics, but many mass spectrometry imaging platforms are commercially available. With greater harmonization across platforms and rigorous quality control, greater adoption of these platforms will undoubtedly provide major insights in complex diseases. Semin Nephrol 36:x-xx © 20xx Elsevier Inc. All rights reserved.

Keywords:  Mass spectrometry; metabolomics; multimodal analyses; proteomics; spatial omics

DOI:  https://doi.org/10.1016/j.semnephrol.2025.151583
RSC Adv. 2025 Apr 16. 15(16): 12757-12764

An approach of molecular-fingerprint prediction implementing a GAT.

Chengzhi Deng, Chengli Zhou, Lei Shi, Bingyi Wang.

In the domain of metabolomics, the accurate identification of compounds is paramount. However, this process is hindered by the vast number of metabolites, which poses a significant challenge. In this study, a novel approach to compound identification is proposed, namely a molecular-fingerprint prediction method based on the graph attention network (GAT) model. The method involves the processing of fragmentation-tree data derived from tandem mass spectrometry (MS/MS) data computation and the subsequent processing of fragmentation-tree graph data with a technique inspired by natural language processing. The model is then trained using a 3-layer GAT model and a 2-layer linear layer. The results demonstrate the method's efficacy in molecular-fingerprint prediction, with the prediction of molecular fingerprints from MS/MS spectra exhibiting a high degree of accuracy. Firstly, this model achieves excellent performance in receiver operating characteristic (ROC) and precision-recall curves. The factors that have the most influence on the resultant performance are identified as edge features using different training parameters. Then, better performance is achieved for accuracy and F 1 score in comparison with MetFID. Secondly, the model performance was validated by querying the molecular libraries through methods commonly used in related studies. In the results based on precursor mass querying, the proposed model achieves comparable performance with CFM-ID; in the results based on molecular formula querying, the model achieves better performance than MetFID. This study demonstrates the potential of the GAT model for compound identification tasks and provides directions for further research.

DOI: https://doi.org/10.1039/d5ra00973a
Nat Protoc. 2025 Apr 21.

Direct analysis of biotransformations with mass spectrometry-DiBT-MS.

Ruth Knox, Rachel Smith, Emily E Kempa, Reynard Spiess, Christian Schnepel, Nicholas J Turner, Sabine L Flitsch, Perdita E Barran.

The development and analysis of engineered enzymes is greatly assisted by the use of high-throughput screening to quickly determine the efficacy of biotransformations under various conditions. Ambient ionization, particularly desorption electrospray ionization (DESI), coupled to high-resolution mass spectrometry has the advantages of minimal requirements for sample preparation before analysis, which renders it suitable for high-throughput screening, in which the accurate mass and potentially the tandem mass spectrometry (MS) fingerprint for any given product can be used for identification. We present a protocol that permits the application of this method in routine biotechnology and chemical biology laboratories that are using engineered enzymes (such as imine reductases and carboxylic acid reductases, mentioned herein) to produce target compounds from substrates (quinoline moieties and phenyl(piperazinyl) moieties, respectively). Through the use of DESI's MS imaging capabilities, reaction monitoring can be easily visualized via imaging of selected substrate or product ions in a convenient, user-friendly workflow. We describe here how DESI-MS can be used to directly analyze the activity of biotransformations from crude cell lysate, which we term 'DiBT-MS'. The DiBT-MS method presented here is 10-1,000 times as fast as liquid chromatography-MS, with the full procedure for 96 samples taking ~2 h and consuming far less solvent and sample. Also demonstrated in this protocol is the impact of solvent spray composition on ionization efficiency of the target analyte, the benefits of a nylon membrane slide and the reusability of sample slides in multiple experiments.

DOI: https://doi.org/10.1038/s41596-025-01161-9
Food Res Int. 2025 May;pii: S0963-9969(25)00491-0. [Epub ahead of print]209 116154

Investigation of polyphenol diversity among lentil species (Lens spp.) using mass spectrometry-based metabolomics guided by photodiode array detection.

Randy W Purves, Hamid Khazaei, Fatma M Elessawy, Roger Munro, Bryn O Shurmer, Albert Vandenberg.

  Polyphenol diversity was investigated among seven lentil species, including Lens culinaris (cultivated lentil), L. orientalis, L. tomentosus, L. odemensis, L. lamottei, L. ervoides, and L. nigricans, using photodiode array detection coupled with liquid chromatography - mass spectrometry (LC-MS). Principal component analysis showed that most species grouped individually, except L. tomentosus and L. odemensis, which overlapped. The LC-MS data from both negative and positive electrospray ionization modes were used to identify 85 polyphenols observed in the UV-vis spectra, which included 27 proanthocyanidins, 17 flavonols, 15 flavones, and 12 hydroxybenzoic acids. An untargeted (comprehensive) analysis of the LC-MS data using Compound Discoverer software identified additional polyphenols (231 total), including numerous overlapping proanthocyanidins that contribute to a broad peak in the UV-vis spectra. The software analysis uncovered some notable differences among polyphenol profiles and intensities within the flavones, flavonols, and phenolic acids present in the species. This result indicates natural variation among the lentil wild relatives, which in part, is attributed to structurally isomeric compounds. A hierarchical clustering analysis, and a differential analysis using volcano plots used to look for statistically significant differences in polyphenols, illustrated significantly lower relative levels of polyphenols in L. culinaris compared with the wild types, especially within the proanthocyanidins and flavones. Our results highlight the potential of lentil wild relatives to enhance lentil seed quality.

Keywords:  Crop wild relatives; LC-MS; Lentils; Polyphenols; Proanthocyanidins; Seed quality; Untargeted analysis

DOI:  https://doi.org/10.1016/j.foodres.2025.116154
J Chromatogr Sci. 2025 Mar 27. pii: bmaf019. [Epub ahead of print]63(4):

A Simple and Sensitive LC-MS/MS Method for Determination of Dapagliflozin and Its Major Metabolite in Human Plasma.

Zhijun Liu, Yanru Liu, Zhipeng Wang, Huiping Lu, Xiujing Zhu, Yuxi Bao, Bingyan Cai, Shouhong Gao, Xia Tao, Deduo Xu.

Dapagliflozin (DAPA), an inhibitor of sodium-dependent glucose cotransporter-2, has been created to treat individuals with type 2 diabetes mellitus. A method was developed and validated using high-performance liquid chromatography coupled to tandem mass spectrometry to aid in studying the connection between clinical effectiveness and concentration of DAPA and its primary metabolite dapagliflozin 3-O-glucuronide (D3OG) in human plasma. The two analytes were separated using the Waters XSELECT HSS T3 (2.1 × 100 mm, 3.5 μm; Waters Co., Milford, USA) chromatographic column, with a mobile phase flow rate of 0.3 mL/min. The elution program was performed after protein precipitation with methanol, which only required a 5-min duration. The extraction recovery was from 99.8 to 109% for DAPA and from 101 to 103% for D3OG. Validation of the method for detecting DAPA within the range of 5-50 ng/mL and D3OG within the range of 50-500 ng/mL demonstrated satisfactory inter- and intra-day precision and accuracy. The method was successfully developed and validated, and it was used to measure the levels of DAPA and D3OG in plasma samples from patients with type 2 diabetes mellitus.

DOI: https://doi.org/10.1093/chromsci/bmaf019
J Am Soc Mass Spectrom. 2025 Apr 25.

Maximizing Data Coverage through Eight Sequential Mass Spectrometry Images of a Single Tissue Section.

Erin H Seeley.

Typical mass spectrometry imaging (MSI) experiments involve the collection of data from only one class of molecules per section. However, it is often necessary to collect data from different classes of analytes from the same biopsy, and generally, serial sections are used for additional analyte classes. However, differences will be observed between the cells present in each section, especially if the sections are not immediately serial with each other. In this study, a method is presented that allows for 8 mass spectrometry images to be collected sequentially from the same tissue section, including metabolites in positive and negative mode, lipids in positive and negative mode, N-linked glycans, O-linked N-acetylglucosamine, small intact proteins, and tryptic peptides. The order of data collection allows for washing to be used that removes analytes already detected and enhances the signal of subsequently imaged analytes. The collection of multiple images from the same tissue section enables facile coregistration of multiple data sets for evaluation of co- and differential localization across molecular classes.

DOI: https://doi.org/10.1021/jasms.5c00032
Food Chem. 2025 Apr 16. pii: S0308-8146(25)01618-8. [Epub ahead of print]484 144367

Improving lipidomic coverage for animal matrices by optimizing extraction methods and resuspension solvents based on UHPLC-Q-Exactive Orbitrap MS/MS.

Ying Wang, Xinhui Bi, Xuhui Huang, Dongmei Li, Lei Qin, Yuying Zhang.

  Due to the complexity of animal matrices and the different polarities of lipids, the measurement methods are expected to cover a comprehensive range of lipid species. Three extraction methods (chloroform/methanol, methyl tert-butyl ether, and Soxhlet methods) and three resuspension solvents (methanol, isopropanol/acetonitrile = 1:1, and isopropanol/acetonitrile = 9:1) were used to compare the lipid species, contents, and ionization efficiency of Spanish mackerel and duck muscles. Overall, the chloroform/methanol extraction method was the most effective in capturing a diverse array of lipid species. The redissolution solvent isopropanol/acetonitrile (1,1) was highly efficient for detecting triglycerides, while methanol was effective in detecting diacylglycerols and phospholipids. Optimizing the measurement conditions of lipids with different polarities provides a valuable reference for lipid extraction and structural elucidation in different muscle tissues. Furthermore, it supports the determination of differential markers for animal lipid metabolism studies or food authentication using an untargeted lipidomic approach.

Keywords:  Extraction methods; Ionization efficiency; Lipid species; Resuspension solvents; Structural analysis; Untargeted lipidomics

DOI:  https://doi.org/10.1016/j.foodchem.2025.144367
Chin J Nat Med. 2025 Apr;pii: S1875-5364(25)60852-1. [Epub ahead of print]23(4): 410-420

Mass spectral database-based methodologies for the annotation and discovery of natural products.

Fengyao Yang, Zeyuan Liang, Haoran Zhao, Jiayi Zheng, Lifang Liu, Huipeng Song, Guizhong Xin.

  Natural products (NPs) have long held a significant position in various fields such as medicine, food, agriculture, and materials. The chemical space covered by NPs is extensive but often underexplored. Therefore, high-throughput and efficient methodologies for the annotation and discovery of NPs are desired to address the complexity and diversity of NP-based systems. Mass spectrometry (MS) has emerged as a powerful platform for the annotation and discovery of NPs. MS databases provide vital support for the structural characterization of NPs by integrating extensive mass spectral data and sample information. Additionally, the released annotation methodologies, based on a variety of informatics tools, continuously improve the ability to annotate the structure and properties of compounds. This review examines the current mainstream databases and annotation methodologies, focusing on their advantages and limitations. Prospects for future technological advancements are then discussed in terms of novel applications and research objectives. Through a systematic overview, this review aims to provide valuable insights and a reference for MS-based NPs annotation, thereby promoting the discovery of novel natural entities.

Keywords:  Annotation; Databases; Mass spectrometry; Natural products

DOI:  https://doi.org/10.1016/S1875-5364(25)60852-1
Analyst. 2025 Apr 22.

A multimodal mass spectrometry imaging workflow for ballpoint pen ink analysis and "forgery" detection.

Veronika Tibljas, Simona Francese, Marjory Da Costa Abreu, Robert Bradshaw.

Inks, including ballpoint, gel, fountain pen, laser toner, inkjet, and security inks, have unique chemical formulations and require specific examination methods based on their properties and the surfaces they are deposited on. Despite the increasing use of digital signatures, ballpoint pens are still used for signing legal documents. Common forensic approaches involving visual examination are crucial in forensic analysis due to their non-destructive nature. However, often their effectiveness can rely on the expertise of the forensic analyst. Other common approaches, such as thin layer chromatography (TLC), require sample extraction which is destructive. This work shows a multimodal imaging workflow, including the application (in order of increasing destructiveness) of Desorption Electrospray Ionisation Mass Spectrometry Imaging (DESI MSI) followed by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) enabling comprehensive ink and substrate analysis in situ. It is important to note that this work was performed in preparation for a real casework study submitted to our group for analysis. The application of DESI MSI in negative ion mode and MALDI MSI in positive ion mode in principle allows for detection and mapping of diverse chemical species (e.g. dyes, polymers, etc.), providing a comprehensive understanding of the chemical composition of the sample. Indeed, it is well documented that each ionisation technique can enable targeting of different molecular species. Statistical approaches used to interrogate the data, allows in-depth analysis of ink/substrates. This approach offers a robust and reliable methodology, which can complement or offer additional information for forgery determination in forensic cases, where the results obtained from conventional methodologies may have not been successful.

DOI: https://doi.org/10.1039/d5an00217f