bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–04–20
53 papers selected by
Sofia Costa, Matterworks
  1. Validation of a Sensitive, Simple and High-Throughput UPLC-MS/MS Method for Quantification of Catecholamines and Their Metabolites in Serum and Urine: Application in Clinical Analysis.
  2. An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of cortisone in human serum and plasma.
  3. Quantification of trimethylamine-N-oxide (TMAO) and its main related trimethylammonium-containing compounds in human plasma by LC-MS/MS.
  4. A convenient and rapid LC-MS/MS method for determination of free and liposomal amphotericin B in human plasma by simultaneous separation using SPE.
  5. Quality evaluation of metabolite annotation based on comprehensive simulation of MS/MS data from high-resolution mass spectrometry (HRMS) and similarity scoring.
  6. Quantification of Ivacaftor, Tezacaftor, Elexacaftor, and Lumacaftor and their active metabolites in plasma using UHPLC-MS/MS: Doors open to the application of therapeutic drug monitoring in cystic fibrosis treatment.
  7. Multiscale workflow for the profiling and identification of urinary food bioactives metabolites Part I: Optimizing urine extraction.
  8. Mechanistic Study of the Deuterium Effect in Chromatographic Separation for Chemical-Tagging Metabolomics and Its Application to Biomarker Discovery in Metabolic Dysfunction-Associated Steatohepatitis.
  9. MS1FA: Shiny App for the Annotation of Redundant Features in Untargeted Metabolomics Datasets.
  10. Library Enabling Annotation of Botanical Natural Products (LEAFBot): An Open-Access Library of Mass Spectrometry Fragmentation Spectra for Plant Metabolites.
  11. Bifunctional Tagging through N-Doped Ozonide for Charge Switching and Isomeric Characterization of Glycerophospholipids Using Tandem Mass Spectrometry.
  12. Skylite: Skyline-Based Lipid Isomer Retention Time Evaluation for Lipidomics in Metabolic Dysfunction-Associated Steatohepatitis.
  13. Quantitative Analysis of Muscarinic Antagonist Atropine and Tiotropium in Microvolume Plasma Using Liquid Chromatography-Mass Spectrometry: Application for Pharmacokinetic Studies.
  14. Evaluation of dried blood spot sampling for verification of exposure to chemical threat agents.
  15. Simultaneous determination of four PDE5 inhibitors and metabolites in rat plasma by UPLC-MS/MS.
  16. Spatial Metabolomics and Lipidomics in Kidney Disease.
  17. Revealing Pharmacokinetic Interactions of Tramadol, Tapentadol, and Venlafaxine: A Cutting-Edge Liquid Chromatography-Tandem Mass Spectrometry Analytical Approach in Rat Plasma.
  18. A New UHPLC-UV-MS Method for Detection and Identification of Bioactive Compounds.
  19. Ex Vivo Measurement of Stable Isotope-Labeled Fatty Acid Incorporation Into Phospholipids in Isolated Mice Muscle.
  20. Phoenics: a novel statistical approach for longitudinal metabolomic pathway analysis.
  21. Sterol and Brassinosteroid Hormone Quantification by LC/MS of Picolinyl Ester Derivatives.
  22. Enzymatic digestion of hair increases extraction yields of cortisol: a novel two-dimensional liquid chromatography-tandem mass spectrometry method for hair cortisol analysis.
  23. Spatial Metabolomics and Its Application in Plant Research.
  24. Liquid- and Gas Chromatography with Tandem Mass Spectrometric Technique to Optimize and Validate the Analysis of Pesticides in Honey: a Large Scope, Multi-Class, Multi-Residue Method.
  25. A sensitive method for simultaneous screening exposure to 14 alkylating agents based on tripeptide adducts: an efficient approach for chemical weapon verification and chemical impurity profiling in plasma samples.
  26. Rapid analysis in small-volume urine: Optimizing LC-MS/MS for free glucocorticoids.
  27. Rapid Determination of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Human Urine Based on Polystyrene/Graphene Oxide Packed-Fibers Solid-Phase Extraction Coupled With High-Performance Liquid Chromatography and Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry.
  28. Development of mass spectrometric methods for determination of desoxycorticosterone pivalate and its esterase product in canine serum.
  29. Detection and accurate identification of Mycobacterium species by flow injection tandem mass spectrometry (FIA-MS/MS) analysis of mycolic acids.
  30. Multimodal Mass Spectrometry Imaging in Atlas Building: A Review.
  31. Advances in Sampling and Analytical Techniques for Single-Cell Metabolomics: Exploring Cellular Heterogeneity.
  32. Adopting ambient ionization mass spectrometry into bioanalytical laboratories.
  33. Development, validation and applications of a GC/MS method for the simultaneous determination of 9 amphetamines and 12 NPS analogues in blood and urine.
  34. Archaeal Lipids: Extraction, Separation, and Identification via Natural Product Chemistry Perspective.
  35. Donor-acceptor covalent organic framework nanofilm-based laser desorption/ionization mass spectrometry for rapid and sensitive determination of creatinine in human serum.
  36. Comprehensive Metabolite Profiling in Single-Cell Systems via Dual-Modal MALDI-Mass Spectrometry Imaging.
  37. MEATiCode: A comprehensive proteomic LC-MS/MS method for simultaneous species identification in meat authentication.
  38. Quantitative Analysis of Pyrrolizidine Alkaloids in Food Matrices and Plant-Derived Samples Using UHPLC-MS/MS.
  39. An LC-MS/MS method for the quantification of tobacco-specific carcinogen protein adducts.
  40. Pharmacokinetics and Tissue Distribution Study for 2-(2-Fluorophenyl)-5-Phenyl-7-Alkoxy- [1,2,4]Triazole[1,5-a]Pyrimidine Antiepileptic Compounds in Rats.
  41. Improving MALDI Mass Spectrometry Imaging Performance: Low-Temperature Thermal Evaporation for Controlled Matrix Deposition and Improved Image Quality.
  42. Sensitive determination of chlorpromazine in pharmaceutical formulations and biological fluids using solid phase extraction followed by HPLC-UV and spectrophotometric analysis.
  43. GC-MS and HPLC-FLD for sensitive assay of toxic cyclohexylamine in artificial sweetener tablets and human biological fluids.
  44. Human plasma protein bindings of neonicotinoid insecticides and metabolites.
  45. Recent advancements in the bioactive alkaloids analysis in plant and biological specimen: From the perspective of activity, sample preparation, and analytical method selection.
  46. Rapid Screening of Illicit Drugs from Biofluid via Dried Blood/Urine Spot and Ultrasonic Desorption-Assisted Low-Temperature Arc Plasma Ionization Mass Spectrometry.
  47. A deep learning framework for enhanced mass spectrometry data analysis and biomarker screening.
  48. Preparation of highly concentrated extracts from large volume of urine as the first step in detecting trace amounts of hypnotics in urine collected in drug-facilitated crime cases.
  49. Fourier Transform Data Analysis for Mass Spectra of Small Polymers and Pyrolysates with Accurate-Mass Data and Mass Defect Preprocessing.
  50. Unique methotrexate polyglutamates distributions in peripheral blood mononuclear cells of rheumatoid arthritis patients: Development and validation of a UPLC-MS/MS method.
  51. Comprehensive review of pyrrolizidine alkaloids in bee products: Occurrence, extraction, and analytical methods.
  52. The ERGtools2 package: a toolset for processing and analysing visual electrophysiology data.
  53. Endogenous metabolites in metabolic diseases: pathophysiological roles and therapeutic implications.
  1. J Chromatogr Sci. 2025 Mar 27. pii: bmaf021. [Epub ahead of print]63(4):
    Validation of a Sensitive, Simple and High-Throughput UPLC-MS/MS Method for Quantification of Catecholamines and Their Metabolites in Serum and Urine: Application in Clinical Analysis.
    Wei Wang, Tiebing Liu, Qingyan Li, Enhui Ji, Weizhe Xu, Shi Qiao, Yujing Cui, Boye Li, Haishan Xu.
      A sensitive, simple and high-throughput UPLC-MS/MS method has been validated for the simultaneous quantification of catecholamines and their metabolite levels in serum and urine for clinical applications. The analytes and their isotope-labeled internal standards were extracted using a 96-well solid-phase extraction cartridge and then separated on an HSS PFP column with a 4-min gradient elution. The linear ranges were 10 ~ 5000 pg/mL for dopamine (DA), epinephrine (E), metanephrine (MN) and normetanephrine (NMN), 2 ~ 5000 pg/mL for norepinephrine (NE), and 2 ~ 2000 pg/mL for 3-methoxytyramine (3-MT). The limits of quantification were 10 pg/mL for DA and E, 5 pg/mL for MN and NMN, 2 pg/mL for 3-MT, and 20 pg/mL for NE. The accuracy was excellent with relative bias all within 10%, and the intra-day and inter-day precision values were also within the tolerance range (RSD < 15%), and the recovery was in the range of 86.0-107.7% with RSD < 15%. After correction using IS, no significant matrix effects were observed. Moreover, the discrepancies in the analyte levels between plasma and serum were investigated for the first time. The analyte levels in the two biological matrices exhibited a significant correlation (P < 0.001) and significant differences (P < 0.001).
    DOI:  https://doi.org/10.1093/chromsci/bmaf021
  2. Clin Chem Lab Med. 2025 Apr 17.
    An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of cortisone in human serum and plasma.
    Myriam Ott, Neeraj Singh, Marie Kubicova, Friederike Bauland, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Christian Geletneky, Judith Taibon.
       OBJECTIVES: Cortisone is an inert precursor/metabolite of the potent steroid hormone cortisol. Measurement of serum cortisone levels and the cortisol-cortisone ratio can be useful for the diagnosis of dysfunction in the regulation of cortisol levels (i.e., severe and subtle apparent mineralocorticoid excess, low-renin primary aldosteronism). Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) to quantify cortisone in human serum/plasma was developed and validated.
    METHODS: Quantitative nuclear magnetic resonance (qNMR) was utilized to assign absolute content (g/g) and SI-traceability to reference materials used as primary calibrators. A supported liquid extraction sample preparation protocol as well as a two-dimensional heart-cut LC approach for LC-MS/MS analysis were employed to mitigate matrix effects and prevent co-elution of interferences. Selectivity was determined by analyzing a matrix sample containing the analyte, the internal standard and six potential interferents. A post-column infusion experiment and a comparison of standard line slopes were performed to evaluate matrix effects. An extensive protocol over five days was applied to determine precision, accuracy and trueness. Measurement uncertainty (MU) was evaluated in compliance with current guidelines.
    RESULTS: This RMP is suitable for analyzing cortisone within the 0.0800-120 ng/mL (0.222-333 nmol/L) range, demonstrating selectivity, sensitivity and matrix independence. Intermediate precision was ≤3.4 %, repeatability was ≤2.9 % across all concentration levels and relative mean bias ranged from -3.7 to 2.8 % across all tested matrices and concentrations. Expanded MU (k=2) for target value assignment (n=6) ranged from 2.1 to 5.5 %, irrespective of concentration or sample type.
    CONCLUSIONS: This RMP allows for accurate and reproducible determination of cortisone in human serum and plasma. Implementation of this method supports routine assay standardization and patient sample measurement with confirmed traceability.
    Keywords:  SI units; cortisone; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability
    DOI:  https://doi.org/10.1515/cclm-2024-1478
  3. Clin Chim Acta. 2025 Apr 11. pii: S0009-8981(25)00173-1. [Epub ahead of print]573 120294
    Quantification of trimethylamine-N-oxide (TMAO) and its main related trimethylammonium-containing compounds in human plasma by LC-MS/MS.
    Salvatore Sotgia.
       BACKGROUND: Trimethylammonium-containing compounds, including choline (CHOL), carnitine (CAR), trimethylglycine (TMG), ergothioneine (ERT), Nε,Nε,Nε-trimethyllysine (TML), γ-butyrobetaine (gBB), and dimethylglycine (DMG) contribute to trimethylamine N-oxide (TMAO) production, a metabolite linked to cardiovascular, renal, and metabolic diseases. An LC-MS/MS method has been established for their simultaneous measurement in human plasma, as an accurate quantification of TMAO and its precursors is crucial for understanding its clinical relevance.
    METHODS: Blood samples from ten healthy male volunteers were processed using acetonitrile protein precipitation. Analysis was performed using a HILIC column and an isocratic methanol-formic acid mobile phase, achieving a total run time of less than 6 min. Linearity was adequate for all analytes (R2 > 0.995), with mean intra- and inter-assay variation coefficients of 2.88 % and 4.23 %, respectively. Recoveries ranged from 95 % to 101 %, limits of detection from 0.009 to 0.068 µmol/L, and limits of quantification from 0.031 to 0.187 µmol/L. Plasma mean concentrations were 3.18 ± 0.73 µmol/L for TMAO, 3.99 ± 0.65 µmol/L for DMG, 9.84 ± 2.08 µmol/L for CHOL, 24.22 ± 6.19 µmol/L for TMG, 0.54 ± 0.22 µmol/L for gBB, 57.29 ± 8.89 µmol/L for CAR, 1.10 ± 0.42 µmol/L for ERT, and 0.40 ± 0.11 µmol/L for TML. Significant inter-individual variability (mean RSD% of 26 %) was observed.
    CONCLUSION: The developed LC-MS/MS method enables rapid, sensitive, and selective quantification of TMAO and its precursors in human plasma. The analytical performance supports its application in clinical and metabolomic studies, contributing to a better understanding the role of TMAO in disease states.
    Keywords:  Clinical diagnostics; LC-MS/MS analysis; Metabolomics; TMAO quantification; Trimethylammonium-containing compounds
    DOI:  https://doi.org/10.1016/j.cca.2025.120294
  4. J Pharm Biomed Anal. 2025 Apr 10. pii: S0731-7085(25)00225-0. [Epub ahead of print]262 116884
    A convenient and rapid LC-MS/MS method for determination of free and liposomal amphotericin B in human plasma by simultaneous separation using SPE.
    Yi Jin, Bowen Wu, Yuting Gong, Huanyu Wei, Ruihan Ma, Yue Wang, Min Yuan, Haiyan Xu.
      Effective separation and specific detection of free and encapsulated drugs in bio-samples are critical to the pharmacokinetic investigation of liposomal medicine. In this study, a simple, convenient, reliable, and selective SPE separation method coupled with sensitive LC-MS/MS technique was developed and fully validated for the detection of liposomal amphotericin B (L-AMB) and non-liposomal amphotericin B (F-AMB) in human plasma. The simultaneous separation between L-AMB and F-AMB in plasma were realized using Oasis HLB SPE cartridge. L-AMB was collected in the aqueous eluate and F-AMB was eluted from the cartridge by methanol containing 1 % formic acid. The analyte and internal standard (natamycin) were separated on a ZORBAX Eclise XDB C18 column (2.1 × 50 mm, 3.5 μm) with gradient elution at a flow rate of 0.5 mL/min, employing a mobile phase that consisted of methanol (0.1 % formic acid) and 5 mM ammonium acetate solution (0.1 % formic acid). Mass spectrometry detection was performed in positive ion mode with electrospray ionization (ESI) interface by multiple reaction monitoring (MRM) method. The linearity range was 200-50000 ng/mL for L-AMB and 10.0-1600 ng/mL for F-AMB. A series of cross quality control samples was adopted to verify the selectivity, stability, and reproducibly of the quantification. Using our methodology, we quantified F-AMB and L-AMB without mutual interferences and obtained excellent incurred sample reanalysis (ISR) results for both analytes. The method was also successfully applied to a clinical study in healthy volunteers.
    Keywords:  Amphotericin B; Free drug; LC-MS/MS; Liposomal drug; Separation
    DOI:  https://doi.org/10.1016/j.jpba.2025.116884
  5. Anal Bioanal Chem. 2025 Apr 18.
    Quality evaluation of metabolite annotation based on comprehensive simulation of MS/MS data from high-resolution mass spectrometry (HRMS) and similarity scoring.
    Yingjiao Shi, Ji Yang, Qianxu Yang, Yipeng Zhang, Zhongda Zeng.
      Metabolite annotation is a critical step in discovery metabolomics, but remains a significant challenge. In this study, the accuracy of metabolite annotation was systematically evaluated by leveraging the proposed strategies for simulation of tandem mass spectrometry (MS/MS) data from high-resolution mass spectrometry (HRMS) and then construction of a large-scale virtual database. Furthermore, various similarity scoring methods were comprehensively compared to assess the performance for annotation. First, three key characteristics that are essential for simulating MS/MS spectra to closely resemble experimental data were identified: (i) the number of mass-to-charge ratio (m/z) features, (ii) the differences between neighboring m/z values, and (iii) the intensity distribution of MS/MS features. These factors were employed to generate representative MS/MS spectra for subsequent study. A meticulously designed virtual MS/MS database was constructed to facilitate accurate annotation assessment, which covered over 100,000 metabolites with diverse structural similarities and differences. To evaluate annotation quality, two simulation strategies on the basis of strong and weak data inference were respectively proposed to replicate MS/MS spectra for unknown metabolites. These simulated spectra were then compared with the virtual database, which provided insights into the expected variations in experimental MS/MS data. Furthermore, eight similarity evaluation methods, including entropy similarity (ES) and weighted dot product (W/DP) algorithms, were rigorously evaluated for their effectiveness in metabolite annotation. The results revealed that some methods, such as ES, exhibited strong resistance to interference and broad adaptability across different MS/MS patterns, whereas others selectively yielded reliable outcomes under specific conditions. This study provided a systematic framework for quality evaluation in metabolite annotation and offered strategies to mitigate false-positive identifications. The findings held great significance for advancing metabolomics research and further improving annotation reliability in complex biological samples.
    Keywords:  Discovery metabolomics; False-positive metabolite annotation; High-resolution mass spectrometry; MS/MS data simulation; Virtual database
    DOI:  https://doi.org/10.1007/s00216-025-05847-7
  6. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Apr 14. pii: S1570-0232(25)00156-4. [Epub ahead of print]1258 124604
    Quantification of Ivacaftor, Tezacaftor, Elexacaftor, and Lumacaftor and their active metabolites in plasma using UHPLC-MS/MS: Doors open to the application of therapeutic drug monitoring in cystic fibrosis treatment.
    A Mireille A Wessels, Femke A Elzinga, Gerard H Koppelman, Onno W Akkerman, Paola Mian, Daan J Touw.
      An ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to quantify the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, tezacaftor, elexacaftor, and lumacaftor and their active metabolites hydroxymethyl ivacaftor, tezacaftor M1, and N-desmethylelexacaftor in human EDTA plasma. The analytical method utilized protein precipitation with stable isotope dilution for sample preparation, facilitating a simple and rapid assay, with a total runtime of only 2.1 min. Separation of the seven components and stable isotope-labeled internal standards was achieved on a C18 column, followed by detection using a tandem quadrupole mass spectrometer. Validation of the method was conducted in accordance with the "Bioanalytical Method Validation Guidance for Industry," of the Food and Drug Administration and with European Medicines Agency's "Guidance on bioanalytical method validation". The assay covers concentrations ranging from 0.010 to 10 mg/L for ivacaftor, hydroxymethyl ivacaftor and N-desmethylelexacaftor, from 0.025 to 25 mg/L for elexacaftor and tezacaftor, from 0.050 to 50 mg/L for tezacaftor M1 and from 0.100 to 100 mg/L for lumacaftor, using a sample volume of 10 μL. Matrix comparison confirmed the applicability of the assay to human serum and heparin plasma. Stability experiments indicated stability of the CFTR modulators in EDTA plasma over ten days under different conditions. At room temperature, all seven components remained stable for eight days and for ten days in the refrigerator in EDTA plasma and in EDTA whole blood. All seven components were stable in EDTA plasma for ten days in the autosampler after sample preparation and through four freeze-thaw cycles. The developed assay was applied in routine TDM analysis to investigate exposure to elexacaftor, tezacaftor, ivacaftor and their metabolites in people with CF undergoing treatment with Kaftrio®.
    Keywords:  Cystic fibrosis; Elexacaftor; Ivacaftor; Lumacaftor; Tezacaftor; UHPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124604
  7. Anal Chim Acta. 2025 Jun 01. pii: S0003-2670(25)00341-1. [Epub ahead of print]1353 343947
    Multiscale workflow for the profiling and identification of urinary food bioactives metabolites Part I: Optimizing urine extraction.
    Evangelos Kalampokis, Theodora Nikou, Stavros Beteinakis, Maria Halabalaki.
       BACKGROUND: The relationship between diet, human health, and disease prevention is well-established, with food bioactive compounds (FBs) widely recognized for their beneficial effects. Metabolism is key in transforming precursor FBs molecules and facilitating their circulation in the human body. Urine has proven to be a valuable biofluid for monitoring dietary exposure. However, the low concentrations of FBs metabolites, their chemical variability, and the lack of appropriate reference standards present challenges in metabolite identification. To address these challenges, developing urine preparation methods for scalable metabolite isolation and unambiguous structure elucidation could significantly improve the coverage and accurate annotation of urine metabolites.
    RESULTS: Urine samples were collected from a healthy volunteer after hydroxytyrosol (HT) supplementation. Traditional urine pretreatment protocols, such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE), were tested alongside enrichment methods using resins (XAD-4, XAD-7, ion-exchange). Extracts were analyzed in parallel using HPLC-DAD/ELSD, UPLC-HRMS, and NMR to assess profiles and annotate metabolites. Methods were evaluated based on extraction yield, metabolite chemical and biochemical diversity, metabolite coverage, selectivity, as well as cost, ease and time. The most promising protocols were further tested on a larger scale. Among the methods evaluated, XAD-7 resin and LLE (Urine/EtOAc 1:3) showed the best performance. Furthermore, detailed identification of metabolites (endogenous and exogenous) per protocol was performed using LC-HRMS/MS and NMR. Additionally, investigation of each protocol performance in respect to the biochemical pathway in which metabolites are implicated was assessed.
    SIGNIFICANCE: The suggested workflow is compatible with both profiling and isolation set-ups and could provide essential insights into urine metabolome and FBs biotransformation. It ensures confident identification and high coverage of metabolites, providing more complete and accurate interpretation of metabolism studies' results and, therefore, valuable input in profiling approaches towards the role of diet on human health.
    Keywords:  Food bioactives; High-scale extraction; Metabolic profiling; Metabolism; Metabolite identification; Sample preparation; Urine
    DOI:  https://doi.org/10.1016/j.aca.2025.343947
  8. Anal Chem. 2025 Apr 15.
    Mechanistic Study of the Deuterium Effect in Chromatographic Separation for Chemical-Tagging Metabolomics and Its Application to Biomarker Discovery in Metabolic Dysfunction-Associated Steatohepatitis.
    Yugo Akioka, Tomoya Higuchi, Takahiro Takayama, Mayuko Ichimura-Shimizu, Koichi Tsuneyama, Koichi Inoue.
      Over the past decade, numerous metabolomics techniques have been developed using liquid chromatography-mass spectrometry (LC-MS). These methodologies have yielded significant findings and facilitated the identification of biomarkers. Among these, chemical-tagging methodologies combined with isotope surrogate tags have garnered considerable attention as a leading approach. Chemical-tagging reduces labor and costs by eliminating the need for internal standard preparation. However, the chromatographic deuterium effect (CDE) has persisted as a significant challenge. CDE poses a risk of data misinterpretation in metabolomics due to potential differences in matrix effects. Although the CDE mechanism has been partially elucidated, it has primarily been attributed to differences in hydrophobicity. A detailed understanding of CDE mechanisms would be valuable for designing chemical tags and optimizing liquid chromatography (LC) conditions. Moreover, emphasizing the CDE could aid in the separation and purification of deuterated compounds. In this study, we investigated the mechanistic basis of the CDE. Initially, four chromatography columns with different separation modes─octadecyl, octadecyl with a positively charged surface, biphenyl, and pentafluorophenyl (PFP) groups─were evaluated based on retention differences between 1H- and 2H6-labeled chemically tagged metabolites. Among these, the PFP column demonstrated the most effective reduction of the CDE, suggesting that electronic interactions with fluorine stabilized 2H-labeled metabolites. Further optimization using the PFP column showed its efficacy in reducing the level of CDE in human serum samples. Finally, the optimized approach was successfully applied to global metabolomics analysis of serum from a mouse model of metabolic dysfunction-associated steatohepatitis.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00289
  9. Bioinformatics. 2025 Apr 15. pii: btaf161. [Epub ahead of print]
    MS1FA: Shiny App for the Annotation of Redundant Features in Untargeted Metabolomics Datasets.
    Ruibing Shi, Frank Klawonn, Mark Brönstrup, Raimo Franke.
       MOTIVATION: Untargeted metabolomics, the comprehensive analysis of small molecules in biological systems, has become an invaluable tool for understanding physiology and metabolism. However, the annotation of metabolomic data is often confounded by the presence of redundant features, which can arise from e.g. multimerization, in-source fragments (ISFs), and adducts.
    RESULTS: MS1FA uniquely integrates all major annotation approaches for redundant features within a single interactive platform. It combines correlation-based grouping with reliable ISF annotation using MS2 data and operates with MS1 data only, MS2 data only, or both. Additionally, it offers a distinctive method for grouping features based on relational criteria. As the only web-based platform with these capabilities, MS1FA provides easy access and allows users to explore and annotate the feature table interactively, with options to download the results.
    AVAILABILITY: MS1FA is freely accessible at https://ms1fa.helmholtz-hzi.de. The source code and data are available at https://github.com/RuibingS/MS1FA_RShiny_dashboard and are archived with the DOI 10.5281/zenodo.15118962.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btaf161
  10. J Am Soc Mass Spectrom. 2025 Apr 18.
    Library Enabling Annotation of Botanical Natural Products (LEAFBot): An Open-Access Library of Mass Spectrometry Fragmentation Spectra for Plant Metabolites.
    Victoria M Anderson, Madhusha M Ranaweera, Alan K Jarmusch, Ashley E Shay, Daniel A Todd, Nadja B Cech, Joshua J Kellogg.
      Many existing mass spectral libraries focus on human or microbially derived molecules. Few plant-specific MS2 databases exist, making annotation of botanical samples difficult. To fill this gap in mass spectrometry data availability, the Library Enabling Annotation of Botanical Natural Products (LEAFBot) was constructed. Using a flow injection mass spectrometry method that allowed for rapid throughput data collection, the MS2 spectra of >300 pure botanical secondary metabolites were experimentally measured and complied into a single library housed in the Global Natural Products Social Molecular Networking (GNPS) spectral database. Of these compounds, over 20% were not present in the existing GNPS database, and 11% were not present in any of three main mass spectral databases (GNPS, Metlin, and MassBank). Additionally, LEAFBot contains a wider range of adducts compared to other plant-based mass spectral libraries, enabling more effective annotation of unknown features. The LEAFBot database represents a new resource to the mass spectrometry and metabolomics community seeking to characterize plant-based samples. The possibility of searching against a taxonomically specific library decreases the likelihood of false positives in database searches, and the ease of adding new spectra, following procedures outlined herein, will enable community-lead expansion of the database.
    DOI:  https://doi.org/10.1021/jasms.5c00038
  11. Anal Chem. 2025 Apr 17.
    Bifunctional Tagging through N-Doped Ozonide for Charge Switching and Isomeric Characterization of Glycerophospholipids Using Tandem Mass Spectrometry.
    Chia-Lung Tsai, Xi Chen, Ramidi G Reddy, Xin Yan.
      Glycerophospholipids (GPLs) are structurally diverse biomolecules that play crucial roles in cellular membranes, signaling, and metabolism. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been widely used for GPL identification due to its high sensitivity and specificity. However, this method often falls short in distinguishing isomeric lipids, such as those differing in the positions of carbon-carbon double bonds. Additionally, the ion types naturally generated during ESI are not always optimal for lipid detection and identification. In this work, we introduce a novel bifunctional tag, nitrophenyl pyrazole (DNPZ), which reacts with double bonds in lipids to form N-doped ozonides. In-situ tandem MS analysis of these modified lipids enables simultaneous identification of double-bond positional isomers and charge switching, facilitating the acquisition of comprehensive structural information. Our findings demonstrate that this approach significantly improves ionization efficiency of GPLs in negative ion mode and provides detailed insights into fatty acyl chain compositions and double-bond positions in GPLs. We have demonstrated that this method allows for the characterization of various lipid classes, lipids with multiple double bonds as well as polar lipid extracts from complex biological samples without the need for authentic lipid reference standards.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00443
  12. Anal Chem. 2025 Apr 14.
    Skylite: Skyline-Based Lipid Isomer Retention Time Evaluation for Lipidomics in Metabolic Dysfunction-Associated Steatohepatitis.
    Jan Philipp Menzel, Fabienne E Birrer, Deborah Stroka, Mojgan Masoodi.
      Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent liver disorder worldwide and can progress to steatohepatitis. Elevated de novo lipogenesis (DNL) is a key contributor to hepatic steatosis. Fatty acid (FA) desaturation produces several unsaturated lipid isomers that are structurally very similar but have diverse biological functions. However, due to their structural similarity, many conventional mass spectrometry approaches cannot detect such metabolic alterations. Thus, we introduce the Skylite (Skyline-based lipid isomer retention time evaluation) workflow using conventional liquid chromatography-mass spectrometry (LC-MS) to identify important isomer features. Retention times of isomeric phosphatidylcholines are compared with the well-characterized human plasma reference standard NIST 1950. Retention time trends correlate well with fixed-charge derivatized FA in liquid chromatography and ozone-induced dissociation mass spectrometry data. The interpretation is supported by double bond diagnostic fragments in LC-MS/MS experiments of epoxidized hydrolyzed fatty acids. We investigate hepatic lipid profiles, focusing on esterified fatty acids in two mouse models of metabolic dysfunction-associated steatohepatitis (MASH). Out of 37 phosphatidylcholine sum compositions, the workflow identifies 123 lipid features. Importantly, CCl4-induced and melanocortin-4 receptor knockout mice on a western diet (WD) have significantly higher levels of mead acid, branched-chain fatty acid, and n-7 PUFA incorporated into phosphatidylcholines. While the MASH mouse liver tissues contain notable amounts of n-7 PUFA, no n-10 PUFA were detected, potentially indicating a unique desaturation pattern. The screening for altered lipid isomer profiles bridges the gap between high-throughput analyses and specialized structure-resolved techniques.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06503
  13. J Anal Methods Chem. 2025 ;2025 9923229
    Quantitative Analysis of Muscarinic Antagonist Atropine and Tiotropium in Microvolume Plasma Using Liquid Chromatography-Mass Spectrometry: Application for Pharmacokinetic Studies.
    Marilène Trancart, Mylène Penot, Gwladys Meesemaecker, Romain Boffy, Anne-Sophie Hanak, André-Guilhem Calas, Nicolas Taudon.
      Despite the availability of current resources, the development of medical countermeasures remains crucial in combatting the threat posed by chemical warfare agents, such as organophosphorus compounds (OPs), which are toxic nerve agents requiring rapid medical intervention. Within the available therapeutic arsenal, muscarinic antagonists such as atropine are administered to mitigate the effects of excessive cholinergic system stimulation, which leads to respiratory tract obstruction due to hypersecretions and bronchoconstriction. Tiotropium, an FDA-approved bronchodilator, acts as a muscarinic receptor antagonist and could, therefore, serve as a potential alternative. To assess its potential efficacy in attenuating OP-induced respiratory effects in a murine intoxication model, it was necessary to first characterize its pharmacokinetic properties. A liquid chromatography-mass spectrometry method was developed and validated following ICH M10 guidelines for the quantification of atropine and tiotropium in 10 μL of plasma. The sample pretreatment procedure involved solid-phase extraction. Chromatographic separation was achieved using a fully porous sub 2 μm C18 column. The analysis was completed in just 4 min, with analytes identified and quantified using two selected reaction monitoring transitions. The mean extraction recoveries exceeded 90% for both drugs, and no matrix effect was observed. The lower limits of quantification were 0.5 ng/mL for tiotropium and 1.0 ng/mL for atropine, with an upper limit of 1000 ng/mL. The signal-to-concentration ratio demonstrated recoveries of back-calculated concentrations ranging from 94% to 108% (relative standard deviation (RSD) < 9.0%). Within-run and between-run precisions were both below 8% with accuracies ranging from 87% to 110%. This highly specific and sensitive method has proven useful for analyzing samples from pharmacokinetic studies conducted in mice. Following intraperitoneal administration, the AUC for tiotropium was approximately twice that of atropine, while its Tmax was half as long (4.9 vs. 8.2 min). The terminal half-lives were approximately 7.5 min for tiotropium and 9.8 min for atropine.
    Keywords:  LC-MS/MS; atropine; medical countermeasure; organophosphorus compounds; pharmacokinetics; plasma; tiotropium
    DOI:  https://doi.org/10.1155/jamc/9923229
  14. Forensic Toxicol. 2025 Apr 15.
    Evaluation of dried blood spot sampling for verification of exposure to chemical threat agents.
    Katie A Walker, Trinity K Rudd, Justin N Vignola, Thomas M McClymont, Noah D Roberts, Kevin Laitipaya, Robert C diTargiani.
       PURPOSE: Exposure to chemical threat agents (CTAs), including nerve agents, the vesicating agent sulfur mustard, and opioids, remains a significant threat to warfighter and civilian populations. Definitive analytical methods to verify exposure to CTAs require shipping refrigerated or frozen biomedical samples to reference laboratories for analysis. Logistical and financial burdens arise as the transport of biomedical samples is subject to strict restrictions and complex packaging, which, if done incorrectly, can lead to sample deterioration. The use of dried blood spot (DBS) sampling could provide operational improvements for collecting, storing, and shipping important forensic samples. Therefore, this effort focuses on developing DBS techniques with Mitra® 30-µL volumetric absorptive microsampling (VAMS®) devices for use in CTA exposure verification.
    METHODS: VAMS® devices were loaded and dried with human whole blood that was exposed to the metabolites pinacolyl methylphosphonic acid (PMPA), ethyl methylphosphonic acid (EMPA), 1,1'sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE), norfentanyl, norcarfentanil, norsufentanil, and norlofentanil. Following extraction from the VAMS® devices, metabolites were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methods were validated for performance by assessing sensitivity, precision, accuracy, and recovery.
    RESULTS: These methods were sensitive to 1 ng/mL for SBMSE, 0.5 ng/mL for PMPA, EMPA, and norfentanyl; 0.1 ng/mL for norlofentanil, and 0.05 ng/mL for norsufentanil and norcarfentanil. All methods met acceptable precision and accuracy criteria with favorable recovery.
    CONCLUSIONS: These results demonstrated the utility of VAMS® in stabilizing human whole blood and show promise as an improved collection method for verification of exposure to various CTAs.
    Keywords:  Dried blood spot; Liquid chromatography; Mass spectrometry; Nerve agent; Opioid; Sulfur mustard
    DOI:  https://doi.org/10.1007/s11419-025-00721-8
  15. J Pharm Biomed Anal. 2025 Apr 09. pii: S0731-7085(25)00224-9. [Epub ahead of print]262 116883
    Simultaneous determination of four PDE5 inhibitors and metabolites in rat plasma by UPLC-MS/MS.
    Xiaonan Xia, Zhe Song, Weiwen He, Zhou Qiao, Fanglin Wang, Shaoyuan Li.
      Herein, we developed a sensitive UPLC-MS/MS method for the simultaneous determination of four PDE5 inhibitor and twelve metabolites in rat plasma. A total of 16 chemicals were separated on gradient elution with mobile phase A (0.1 % formate acetonitrile) and mobile phase B (0.1 % formate and 2 mmol/L ammonium formate aqueous solution) on an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) after protein precipitation, followed by the detection on the positive ion mode of electrospray ion source (ESI) via dynamic multi-reaction monitoring mode (MRM). The method provided good linearity over the range of 0.25-20 ng/mL for all compounds with correlation coefficients R greater than 0.99. The recoveries were higher than 75.5 %, the matrix factors were 2.1 %-20.4 %. The precision, accuracy and stability were also within acceptable criteria. A rat model was established to investigate the metabolic profile of Sildenafil, Vardenafil, Acetildenafil and Tadalafil. Blood samples from the experimental group were analyzed using ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Notably, desmethyltadalafil and methyl-desmethyltadalafil were not detected in the plasma. However, their glucuronide conjugates, desmethyltadalafil glucuronide and methyl-desmethyltadalafil glucuronide, were identified and characterized via tandem mass spectrometry (MS/MS). At present, cases of poisoning or even death due to overdose of phosphodiesterase type 5 (PDE5) inhibitors are often encountered in the practice of forensic science. Few studies on PDE5 inhibitors have been reported in forensic science toxicology identification, and the existing detection methods cannot meet the need for simultaneous and rapid analysis of multiple trace PDE5 inhibitors in vivo. Therefore, it is of great importance to establish and optimize the detection methods for PDE5 inhibitors drugs in biological samples. The significance of this method is to provide a reference for the determination of PDE5 inhibitors poisoning cases.
    Keywords:  Determination; PDE5 Inhibitor; Sildenafil; Tadalafil; UPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.jpba.2025.116883
  16. Semin Nephrol. 2025 Apr 15. pii: S0270-9295(25)00019-1. [Epub ahead of print] 151582
    Spatial Metabolomics and Lipidomics in Kidney Disease.
    Brittney L Gorman, Jessica K Lukowski.
      Kidney disease is a global health issue that affects over 850 million people, and early detection is key to preventing severe disease and complications. Kidney diseases are associated with complex and dysregulation of lipid metabolism. Spatial metabolomics through mass spectrometry imaging (MSI) enables spatial mapping of the lipids in tissue and includes a variety of techniques that can be used to image lipids. In the kidney, MSI studies often seek to resolve individual functional tissue units such as glomeruli and proximal tubules. Several different MSI techniques, such as matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI), have been used to characterize lipids and small molecules in chronic kidney disease, acute kidney injury, genetic kidney disease, and cancer. In this review we provide several examples of how spatial metabolomics data can provide critical information concerning the localization of changes in various disease states. Additionally, when combined with pathology, transcriptomics, or proteomics, the metabolomic changes can illuminate underlying mechanisms and provide new clinical insights into disease mechanisms. Semin Nephrol 36:x-xx © 20xx Elsevier Inc. All rights reserved.
    Keywords:  Kidney; MALDI; lipidomics; mass spectrometry imaging; metabolomics
    DOI:  https://doi.org/10.1016/j.semnephrol.2025.151582
  17. ACS Pharmacol Transl Sci. 2025 Apr 11. 8(4): 1116-1128
    Revealing Pharmacokinetic Interactions of Tramadol, Tapentadol, and Venlafaxine: A Cutting-Edge Liquid Chromatography-Tandem Mass Spectrometry Analytical Approach in Rat Plasma.
    Hala M Heneedak, Khaled M Darwish, Samia M Mostafa, Mohamed Saleh Elgawish.
      The detection and quantification of opioid analgesics adulterated with serotonin reuptake inhibitors are critical in forensic toxicology due to their significant clinical and legal implications. This study focuses on developing and validating a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of tramadol (TMD), tapentadol (TAP), and venlafaxine (VEN) in small volumes of rat plasma. The method employs a straightforward single-step protein precipitation technique for sample pretreatment. Utilizing a Shim-pack Velox SP-C18 column (2.7 μm, 2.1 × 150 mm, Shimadzu, Japan), a 12 min gradient elution was performed with a mobile phase of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Detection and quantification were achieved in multiple-reaction-monitoring mode, with ion transitions of m/z 264.3 → 58.1 for TMD, m/z 222.3 → 107.1 for TAP, m/z 278.2 → 58.2 for VEN, and m/z 195.5 → 138.1 for caffeine, which served as the internal standard (IS). The method exhibited excellent linearity, with concentration ranges of 1-500 ng/mL for TMD (r 2 = 0.9981), 1-1000 ng/mL for TAP (r 2 = 0.9972), and 1-900 ng/mL for VEN (r 2 = 0.9987) in rat plasma using only 50 μL of sample. This validated method was applied to a pharmacokinetic interaction study, revealing significant drug interactions: the maximum observed plasma concentration (C max) and area under the curve (AUC) for both TMD and VEN decreased. For TMD, C max decreased by 2.58-fold from 276.25 to 106.99, and AUC0-t decreased by 1.4-fold from 3005.8 to 2159.3. For VEN, C max decreased by 2.51-fold from 494 to 191, and AUC0-t decreased by 2.3-fold from 4988 to 2114. However, for TAP, C max increased by 3.4-fold from 138.54 to 471.85, and AUC0-t increased by 2.66-fold from 1060.1 to 2826.8. The concurrent administration likely creates metabolic competition at CYP2D6 and UDP glycosyltransferase enzyme sites, affecting the pharmacokinetic parameters. These findings underscore the importance of further studies to monitor the simultaneous presence of these drugs and their metabolites in plasma, especially when coadministration occurs unintentionally.
    DOI:  https://doi.org/10.1021/acsptsci.4c00722
  18. J AOAC Int. 2025 Apr 18. pii: qsaf039. [Epub ahead of print]
    A New UHPLC-UV-MS Method for Detection and Identification of Bioactive Compounds.
    Adam T Zarth, Ana M Magallanes López.
       BACKGROUND: The development of analytical methods in food science has grown parallel with consumers' concern about food composition. There is desire to better understand the phytochemicals present in foods and feeds. Comprehensive screening of nutrients and bioactive compounds can be challenging due to the vast differences in molecular properties of the compounds.
    OBJECTIVE: To develop a sensitive liquid chromatography-mass spectrometry (LC-MS) analytical method to detect, identify, and quantify hundreds of compounds from a broad range of molecular categories in a single method, from hydrophilic (e.g., organic acids) to hydrophobic (e.g., sterols) nutrients.
    METHOD: The chromatographic separation was tailored for a balanced detection of hydrophilic and hydrophobic compounds, with attention towards the separation of isomeric polyphenols. The chromatographic separation was performed in 45 min on a C18-PFP stationary phase, which provided enhanced resolution of isomers. The method employs two columns in series to improve the resolution, which requires ultra-high-pressure instrumentation (1,000 bar). The addition of an in-line ultraviolet (UV) diode-array detector allowed for spectral profile confirmation of the identities of many targeted and unknown compounds.
    RESULTS: The developed method was applied in this work to measure different types of bioactives recognized to have health benefits: flavonoids, phenolic acids, vitamins, tocochromanols, phytosterols, sugars, organic acids, lipids, amines, nucleic acids, and many other small molecules. Four chocolate products were analyzed as a demonstration of the range of the method.
    CONCLUSIONS: This method has enabled discoveries of new commercial value in the food processing industry by capturing information on a wider range of analytes than previous individual methods, and this data can be leveraged to promote the health and well-being of consumers.
    HIGHLIGHTS: This new LC-UV-MS methodology provides a more comprehensive analysis of a broad range of molecularly diverse bioactive constituents in raw materials and finished products in the food and feed industry.
    DOI:  https://doi.org/10.1093/jaoacint/qsaf039
  19. Bio Protoc. 2025 Apr 05. 15(7): e5257
    Ex Vivo Measurement of Stable Isotope-Labeled Fatty Acid Incorporation Into Phospholipids in Isolated Mice Muscle.
    Tomoki Sato, Shinji Miura.
      With the advancement of liquid chromatography-mass spectrometry (LC-MS/MS), the quantification of glycerophospholipid (PL) molecules has become more accessible, leading to the discovery of numerous enzymes responsible for determining the acyl groups attached to these molecules. Metabolic tracer experiments using radioisotopes and stable isotopes are powerful tools for defining the function of metabolic enzymes and metabolic flux. We have established an ex vivo muscle experimental system using stable isotope-labeled fatty acids to evaluate fatty acid incorporation into PL molecules. Here, we describe a method to incorporate fatty acids with stable isotope labels into excised skeletal muscle and detect the PL molecules containing labeled acyl chains by LC-MS/MS. Key features • Quantify the metabolism of fatty acids into phospholipid acyl chains. • Enable measurements in excised muscle samples. • Assess the effects of genetic recombination of acyltransferases.
    Keywords:  Acyl chain; Free-fatty acid; Liquid chromatography–mass spectrometry; Phospholipid; Skeletal muscle; Stable isotope tracer
    DOI:  https://doi.org/10.21769/BioProtoc.5257
  20. BMC Bioinformatics. 2025 Apr 16. 26(1): 105
    Phoenics: a novel statistical approach for longitudinal metabolomic pathway analysis.
    Camille Guilmineau, Marie Tremblay-Franco, Nathalie Vialaneix, Rémi Servien.
       BACKGROUND: Metabolomics describes the metabolic profile of an organism at a given time by the concentrations of its constituent metabolites. When studied over time, metabolite concentrations can help understand the dynamical evolution of a biological process. However, metabolites are involved into sequences of chemical reactions, called metabolic pathways, related to a given biological function. Accounting for these pathways into statistical methods for metabolomic data is thus a relevant way to directly express results in terms of biological functions and to increase their interpretability.
    METHODS: We propose a new method, phoenics, to perform differential analysis for longitudinal metabolomic data at the pathway level. In short, phoenics proceeds in two steps: First, the matrix of metabolite quantifications is transformed by a dimension reduction approach accounting for pathway information. Then, a mixed linear model is fitted on the transformed data.
    RESULTS: This method was applied to semi-synthetic NMR data and two real NMR datasets assessing the effects of antibiotics and irritable bowel syndrome on feces. Results showed that phoenics properly controls the Type I error rate and has a better ability to detect differential metabolic pathways and to extract new impacted biological functions than alternative methods. The method is implemented in the R package phoenics available on CRAN.
    Keywords:  Longitudinal data; Metabolic pathways; Metabolomics; Mixed model
    DOI:  https://doi.org/10.1186/s12859-025-06118-z
  21. Cold Spring Harb Protoc. 2025 Apr 17.
    Sterol and Brassinosteroid Hormone Quantification by LC/MS of Picolinyl Ester Derivatives.
    Brian P Dilkes, Norman B Best.
      Brassinosteroids are small steroidal hormones that regulate plant growth, differentiation, and defense. They are low abundance in plant tissues and are difficult to assess via mass spectrometry due to poor ionization. In this protocol, we provide a method for the extraction, detection, and quantification of a subset of sterol and brassinosteroid metabolites using a derivatization method to improve ionization during liquid chromatography coupled with mass spectrometry. Multiple reaction monitoring, which is the utilization of metabolite fragments made in the instrument, is used to distinguish the sterols from other metabolites in complex mixtures to allow the simultaneous detection of a wide variety of steroids, including brassinosteroids. In maize, genetic resources have permitted multiple insights into the role of brassinosteroids in growth and development, and the addition of this convenient protocol to quantify their levels in plant tissue will enable a deeper physiological and biochemical understanding.
    DOI:  https://doi.org/10.1101/pdb.prot108646
  22. Anal Bioanal Chem. 2025 Apr 16.
    Enzymatic digestion of hair increases extraction yields of cortisol: a novel two-dimensional liquid chromatography-tandem mass spectrometry method for hair cortisol analysis.
    Dewi van Harskamp, Mariëtte T Ackermans, Wjera V Wickenhagen, Annemieke C Heijboer, Johannes B van Goudoever.
      Hair cortisol concentration is a retrospective, long-term measure of cortisol secretion, often used as a biomarker for chronic stress or suspected cyclic Cushing's syndrome. Various methods for hair cortisol extraction, typically involving intact, minced, or milled hair incubated with methanol or extraction buffers, may not achieve complete extraction. Incomplete extraction can bias results, particularly when comparing hair samples of differing thickness and texture. Additionally, sample-to-sample variation might be caused by inconsistent cutting or grinding of the hair by different technicians. We aimed to achieve complete cortisol extraction through the enzymatic digestion of hair. After digestion and sample clean-up, we analyzed the final extracts using two-dimensional liquid chromatography-mass spectrometry. Complete digestion was observed with an overnight enzymatic reaction. This method demonstrated high reproducibility, with an intra-day precision of 3.6%, and inter-day precision of 6.5% in a homogenized pool. Moreover, duplicate CV% of intact hairlocks ranged from 0.1% to 8.9%. Our protocol does not influence the cortisol concentration. It is less labor-intensive and more consistent than mincing or milling hair. Our extraction yield was higher compared to the commonly used methanol extraction method. Samples extracted with methanol showed, on average, 19% lower cortisol concentrations than those measured in digested aliquots. We have developed and validated a new, superior method for extracting cortisol from hair utilizing enzymatic digestion. This strategy provides a more thorough extraction, greater consistency, and requires less labor than traditional methods.
    Keywords:  Chronic stress; Hair cortisol concentration; Liquid chromatography; Mass spectrometry; Proteinase K for digestion of hair; Retrospective
    DOI:  https://doi.org/10.1007/s00216-025-05878-0
  23. Int J Mol Sci. 2025 Mar 26. pii: 3043. [Epub ahead of print]26(7):
    Spatial Metabolomics and Its Application in Plant Research.
    Rong Li, Fang Wang, Jian Wang.
      Spatial metabolomics, as a frontier technology, is capable of conducting the comprehensive characterization of metabolites within organisms in terms of qualitative, quantitative and positional dimensions, so as to facilitate the visual analysis of biological processes. This paper summarizes the birth and development of spatial metabolomics, explains its differences and advantages from traditional metabolomics and summarizes its application in plant research. In addition, the limitations of spatial metabolomics are summarized and discussed, along with the technological improvement and application innovation of spatial metabolomics, in order to provide reference for the development strategy of spatial metabolomics and its application in plant research.
    Keywords:  mass spectrometry imaging; spatial distribution; spatial metabolome
    DOI:  https://doi.org/10.3390/ijms26073043
  24. J AOAC Int. 2025 Apr 17. pii: qsaf040. [Epub ahead of print]
    Liquid- and Gas Chromatography with Tandem Mass Spectrometric Technique to Optimize and Validate the Analysis of Pesticides in Honey: a Large Scope, Multi-Class, Multi-Residue Method.
    Partha P Choudhury, Sachin C Ekatpure, Abhishek Mandal, Veena Rao Udupi, Kusuma D Krishnamurthy, Nethravati Bheemiah, Seema Shenvi, Kaushik Banerjee.
       BACKGROUND: Honey is a nutrient-rich food item with a complex matrix due to its diverse nutritional components. This complexity poses challenges in the analysis of pesticide residues, affecting accuracy and precision of results. A comprehensive and reliable method is required for the detection and quantification of these contaminants.
    OBJECTIVE: To develop and validate a method for simultaneous analysis of pesticide residues in various honey types using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS).
    METHODS: Honey samples (5 g) were extracted using acetonitrile (10 mL) and water (10 mL) for LC-MS/MS analysis, while ethyl acetate (10 mL) was used for GC-MS/MS analysis. The extract was cleaned using primary secondary amine (PSA) before pesticide residue quantification by LC-MS/MS and GC-MS/MS. The method performance was evaluated based on recoveries (70-120%) and repeatability (RSD <20%) at a limit of quantification of 0.01 mg/kg, in accordance with SANTE/11312/2021 guidelines.
    RESULTS: The developed method demonstrated compliance with regulatory requirements, ensuring reliable determination of pesticide residues in honey samples. A study on matrix variability from 14 different honey samples revealed that each honey type exhibited a unique matrix effect for the targeted pesticides. To minimize this effect, the use of honey type-specific matrix-matched standards is recommended.
    CONCLUSION: The study successfully developed a simple, robust, and high-throughput analytical method for the quantitative determination of pesticide residues in honey. Given its high selectivity, sensitivity, and rugged performance, the method can be implemented in regulatory testing for the analysis of targeted compounds across a wide range of honey matrices.
    HIGHLIGHTS: A method for pesticide residue analysis in honey was developed using LC-MS/MS and GC-MS/MS. The method showed acceptable recoveries (within 70-120%) and repeatability (RSD <20%) at an LOQ of 0.01 mg/kg. Each honey type exhibited a unique matrix effect, emphasizing the need for matrix-matched standards for residue quantifications. The method demonstrated high throughput, selectivity, and sensitivity, making it suitable for regulatory testing.
    DOI:  https://doi.org/10.1093/jaoacint/qsaf040
  25. Anal Bioanal Chem. 2025 Apr 17.
    A sensitive method for simultaneous screening exposure to 14 alkylating agents based on tripeptide adducts: an efficient approach for chemical weapon verification and chemical impurity profiling in plasma samples.
    Bo Chen, Deshen Liang, Huilan Yu, Changcai Liu, Ling Yuan, Fengyun Wang, Shilei Liu.
      For the implementation of the Chemical Weapons Convention, the Organisation for Prohibition of Chemical Weapons (OPCW) designates laboratories for the analysis of chemicals that can be used as chemical warfare agents (CWAs). In these laboratories, analytical methods for detecting CWAs have been developed for environmental and biomedical samples. Protein adducts from exposed biomedical samples are an important type of biomarker for verification of their abuse. Moreover, these adducts could also be applied in chemical impurity profiling studies of biomedical samples. Alkylating agents, as cytotoxicants, can react with Cys34 in human serum albumin, and cysteine-proline-phenylalanine (CPF) tripeptide adducts generated from proteinase K digestion of modified albumin have been used as biomarkers for retrospective detection of exposure to sulfur mustards or nitrogen mustards. In this study, a sensitive screening method for detecting exposure to 14 alkylating agents in one analytical run was established through sample preparation and instrumental detection optimization. Ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-TQ MS) operated in multiple reaction monitoring (MRM) mode and combined with an optimized chromatographic program was used for detecting the 14 corresponding CPF tripeptide adducts. The limits of detection (LODs) are in the range of 0.200-10.0 ng/mL exposure concentrations in human plasma. This method could also allow impurity profiling applicable for potential attribution of sulfur and nitrogen mustard in plasma through retrospective analysis of exposed biomedical samples with impurity compositions as low as 0.1%. This method has potential in clinical diagnosis, CWA verification and forensic identification applications.
    Keywords:  Alkylating agent; Biomarker; Chemical impurity profiling; Nitrogen mustards; Sulfur mustards; Tripeptide adduct
    DOI:  https://doi.org/10.1007/s00216-025-05870-8
  26. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Apr 03. pii: S1570-0232(25)00131-X. [Epub ahead of print]1257 124579
    Rapid analysis in small-volume urine: Optimizing LC-MS/MS for free glucocorticoids.
    Ikumi Endo, Mikiko Tokiya, Kazuhiro Kawamoto, Yasuyuki Maeda, Sudarma Bogahawaththa, Masayoshi Ichiba, Akiko Matsumoto.
      Glucocorticoids, regulators of energy metabolism and immune responses, are known biomarkers of stress response and disease. Downscaling the required sample volumes facilitates large-scale studies and accelerates the generation of valid results. To meet this expectation, we modified our LC-MS/MS method using 100 μL of urine to that using 20 μL, following the same steps as in the original protocol with the addition of cortisone-d8 as an internal standard. Calibration was performed as in the original protocol but using 1/5 diluted standards, resulting in a good linearity of 2-200 ng/mL in the log-log plot, indicating a lower limit of quantitation (LLOQ) of <2 ng/mL from the residue. The coefficients of variation of the matrix factors of the 15 urine samples and water were within 15 % for 10 and 300 ng/mL cortisol and cortisone. The matrix factor of the downscaled method was larger than that of the original method, especially for urine extracts with high specific gravity (up to 2.1-fold), indicating the advantage of the downscaled method; although the theoretical LLOQ is 5-fold with the 1/5 downscaling, the effect on the practical LLOQ may be reduced by the suppressed matrix effect. The current method showed good recovery, accuracy, reproducibility, and agreement with the original method (regression coefficient close to 1.0). After sample freezing, cortisol and cortisone were detected at higher and lower levels, respectively, within 25 %. The downscaled method of urine-free cortisol and cortisone may contribute to the advancement of related research.
    Keywords:  Downscaling; Free cortisol; Free cortisone; LC-MS/MS; Small volume; Urine
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124579
  27. J Sep Sci. 2025 Apr;48(4): e70130
    Rapid Determination of 25-Hydroxyvitamin D3 and 24,25-Dihydroxyvitamin D3 in Human Urine Based on Polystyrene/Graphene Oxide Packed-Fibers Solid-Phase Extraction Coupled With High-Performance Liquid Chromatography and Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry.
    Qing Han, Min Zhou, Xuejun Kang, Guozhe Deng, Feng Guo, Li Geng.
      Vitamin D plays a crucial role in skeletal metabolism and is implicated in various diseases. Several studies have indicated that human vitamin D status can be evaluated through urine hydroxyvitamin D levels, in addition to plasma hydroxyvitamin D concentrations. In this study, we developed a novel method for the detection of trace amounts of 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] in urine. This method is based on high-performance liquid chromatography coupled with atmospheric pressure chemical ion source-tandem mass spectrometry, integrated with packed-fibers solid-phase extraction. The composite polystyrene/graphene oxide (PS/GO) nanofibers were synthesized as the sorbent for concentrating 25(OH)D3 and 24,25(OH)2D3, achieving a twentyfold increase in sensitivity when analyzing human urine samples. Fourier-transform infrared spectroscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy mapping analysis were employed to characterize GO, PS, and PS/GO. The results indicated that the composite nanofibers were successfully synthesized. In addition, we investigated the relationship between the extraction performance of the fibers and the electrospinning process at varying flow rates of spinning solution through morphological studies and Brunauer-Emmett-Teller surface area analysis. During the extraction process, purification, concentration, and desorption were accomplished within a single step. The established method demonstrated excellent sensitivity and efficiency. Under optimal conditions, limits of detection (signal-to-noise ratio = 3) were found to be 0.33 ng/mL for 25(OH)D3 and 0.19 ng/mL for 24,25(OH)2D3; furthermore, linearity was deemed acceptable across urine samples. Recovery rates ranged from 89.5% to 109.7% for 25(OH)D3 and from 90.3% to 103.1% for 24,25(OH)2D3, respectively.
    Keywords:  LC‐MS/MS; nanofibers; solid‐phase extraction; urine; vitamin D3 metabolites
    DOI:  https://doi.org/10.1002/jssc.70130
  28. Toxicol Mech Methods. 2025 Apr 14. 1-13
    Development of mass spectrometric methods for determination of desoxycorticosterone pivalate and its esterase product in canine serum.
    A F Lehner, Justin Zyskowski, J P Buchweitz, D K Langlois.
      Hypoadrenocorticism is a serious condition in dogs that results from autoimmune adrenalitis and depletion of mineralocorticoids and glucocorticoids. Affected dogs respond well to glucocorticoid supplementation and treatment with the synthetic mineralocorticoid desoxycorticosterone pivalate (DOCP). DOCP injected once monthly resolves serum Na/K abnormalities and normalizes water balance, but therapy is expensive. Cost abatement involves prolongation of the 30-day dosage interval or decreasing the 2.2 mg/kg dosage. These approaches are not based on DOCP pharmacokinetics. A full assessment of the practicality of either approach would benefit from understanding drug pharmacokinetics, requiring measurement of DOCP and its esterase product desoxycorticosterone (DOC) in canine serum while avoiding toxic endpoints from overdosing. Mass spectrometric methods were developed including gas chromatography-tandem mass spectrometry of DOCP and DOC-methoxime trimethylsilyl derivatives, an approach sensitive to 2 ng/mL. Greater sensitivity was desired, so liquid chromatography-tandem mass spectrometry (LC-MS/MS) with ESI+ ionization was investigated. Supported liquid extraction was devised for serum with recoveries ∼100%. The LC-MS/MS method was validated for linearity, precision, accuracy and limits of detection (0.029 and 0.019 ng/mL for DOC and DOCP, respectively). A pilot experiment with DOCP-treated hypoadrenocorticism dogs over one-month revealed DOC baseline values as 0.183+/-0.090 ng/mL, which increased to the 1.0 - 2.2 ng/mL range. DOCP was not visible in any samples suggesting 100% conversion. Halving the dosage to 1.1 mg/kg still showed clear increases over the DOC baseline. MS fragmentation involved ring cleavages, dehydrations and double-charged fragments. The methodology was robust and suitable for studying DOC/DOCP pharmacokinetics in future studies of hypoadrenocorticism dogs.
    Keywords:  Addison’s disease; LC-MS/MS; canine hypoadrenocorticism; desoxycorticosterone; desoxycorticosterone pivalate
    DOI:  https://doi.org/10.1080/15376516.2025.2489026
  29. Sci Rep. 2025 Apr 16. 15(1): 13118
    Detection and accurate identification of Mycobacterium species by flow injection tandem mass spectrometry (FIA-MS/MS) analysis of mycolic acids.
    Szewczyk Rafał, Druszczynska Magdalena, Majewski Karol, Szulc Bartłomiej, Kowalski Konrad.
      Mycolic acids, long-chain α-alkyl, β-hydroxy fatty acids, are characteristic for the genus Mycobacterium and play a critical role in the structural integrity and pathogenicity of mycobacterial cell walls. The unique structural diversity of mycolic acids among various Mycobacterium species offers a reliable biomarker for taxonomic differentiation. In this study, we applied flow injection analysis coupled with tandem mass spectrometry (FIA-MS/MS) for a comprehensive profiling of mycolic acids. Our findings demonstrate that the structural diversity of mycolic acids can be effectively utilized to enhance the accuracy, sensitivity, and scalability of mycobacterial identification methods. This technique has a broad applications for the rapid identification of mycobacterial pathogens and determination of drug resistance. Particularly in clinical diagnostics it may lead to improvement of patient management and treatment outcomes. Additionally, the method holds promise for epidemiological surveillance, enabling the tracking of mycobacterial outbreaks, as well as applications in environmental microbiology for the detection and monitoring of mycobacteria in diverse ecosystems.
    Keywords:   Mycobacterium ; Chemotaxonomy; Flow injection tandem mass spectrometry (FIA/MS-MS); Innovative diagnostic methods; Mycolic acids; Tuberculosis
    DOI:  https://doi.org/10.1038/s41598-025-96867-x
  30. Semin Nephrol. 2025 Apr 16. pii: S0270-9295(25)00015-4. [Epub ahead of print] 151578
    Multimodal Mass Spectrometry Imaging in Atlas Building: A Review.
    Kyle A Vanderschoot, Kayle J Bender, Christopher M De Caro, Kelli A Steineman, Elizabeth K Neumann.
      In the era of precision medicine, scientists are creating atlases of the human body to map cells at the molecular level, providing insight into what fundamentally makes each cell different. In these atlas efforts, multimodal imaging techniques that include mass spectrometry imaging (MSI) have revolutionized the way biomolecules, such as lipids, peptides, proteins, and small metabolites, are visualized in the native spatial context of biological tissue. As such, MSI has become a fundamental arm of major cell atlasing efforts, as it can analyze the spatial distribution of hundreds of molecules in diverse sample types. These rich molecular data are then correlated with orthogonal assays, including histologic staining, proteomics, and transcriptomics, to analyze molecular classes that are not traditionally detected by MSI. Additional computational methods enable further examination of the correlations between biomolecular classes and creation of visualizations that serve as a powerful resource for researchers and clinicians trying to understand human health and disease. In this review, we examine modern multimodal imaging methods and how they contribute to precision medicine and the understanding of fundamental disease mechanisms. Semin Nephrol 36:x-xx © 20XX Elsevier Inc. All rights reserved.
    Keywords:  Multimodal imaging; mass spectrometry; multiomics; single-cell analysis; tissue imaging
    DOI:  https://doi.org/10.1016/j.semnephrol.2025.151578
  31. Rapid Commun Mass Spectrom. 2025 Jul 15. 39(13): e10045
    Advances in Sampling and Analytical Techniques for Single-Cell Metabolomics: Exploring Cellular Heterogeneity.
    Xinxin Shen, Fangyuan Zhang, Chunping Tang, Marina Soković, Danijela Mišić, Hongxi Xu, Yang Ye, Jia Liu.
      Single-cell metabolomics is an emerging and powerful technology that uncovers intercellular heterogeneity and reveals microenvironmental dynamics in both physiological and pathological conditions. This technology enables detailed observations of cellular interactions, providing valuable insights into processes such as aging, immune responses, and disease development. Despite significant advances, the need for detailed discussions on sampling and analytical methods in single-cell metabolomics continues to grow, with increasing focus on selecting the most suitable techniques for diverse research objectives. This review addresses these challenges by exploring key sampling and analytical strategies used in single-cell metabolomics. We focus on three main approaches: the capture and isolation of specific cell types, the precise aspiration of individual cells, and in situ mass spectrometry imaging. These methods are critically assessed to highlight strategies for achieving accurate metabolite detection at the single-cell level across diverse research applications.
    Keywords:  cell isolation; mass spectrometry imaging; metabolite detection; sampling techniques; single‐cell metabolomics
    DOI:  https://doi.org/10.1002/rcm.10045
  32. Bioanalysis. 2025 Apr 12. 1-5
    Adopting ambient ionization mass spectrometry into bioanalytical laboratories.
    Forough Doustkhahvajari, Stephanie Rankin-Turner.
      
    Keywords:  Mass spectrometry; ambient ionization; bioanalytical; biomarkers; clinical; immunoassay; metabolomics
    DOI:  https://doi.org/10.1080/17576180.2025.2490462
  33. Forensic Sci Int. 2025 Apr 09. pii: S0379-0738(25)00107-0. [Epub ahead of print]370 112469
    Development, validation and applications of a GC/MS method for the simultaneous determination of 9 amphetamines and 12 NPS analogues in blood and urine.
    Christoforos Bouzoukas, Panagiota Nikolaou, Sotirios Athanaselis, Artemisia Dona, Chara Spiliopoulou, Ioannis Papoutsis.
      Amphetamine-type stimulants (ATS), synthetic cathinones (SCs) and phenethylamines (PEAs) pose a challenge to toxicology laboratories. Their extensive use and misuse has led to an increase of intoxications and fatal incidents worldwide. So, the need for new analytical methods for their determination in biological samples is crucial for all toxicology laboratories for a better investigation of these cases. The aim of this study was the development and validation of an analytical method for the determination of 9 ATS, 7 SCs and 5 PEAs in whole blood and urine using gas chromatography coupled with mass spectrometry. The samples were pretreated by solid-phase extraction and derivatized with pentafluoropropionic anhydride. Chromatographic separation of the 21 analytes was achieved in less than 11 min. The method was validated according to international guidelines for selectivity, specificity, linearity, limits of detection (LODs) and quantification (LOQs), precision, accuracy and recovery of the method. The LODs of the analytes ranged from 0.70 to 7.0 ng/mL and the values of the LOQs ranged from 2.0 to 20 ng/mL. The linearity of the 21 analytes, according to the group of substances, were 2.0 ng/mL - 0.20 μg/mL, 5.0 ng/mL - 0.50 μg/mL, 10 ng/mL - 0.50 μg/mL and 20 ng/mL - 0.50 μg/mL with R2 values > 0.99. Extraction recoveries were > 80 % for all analytes. Intra and inter day accuracy and precision of the method were within accepted limits. The developed method was applied to post-mortem blood and urine samples of 46 forensic cases sent for toxicological investigation to the Department of Forensic Medicine and Toxicology, School of Medicine, National and Kapodistrian University of Athens. The results of the analyses revealed the presence of methamphetamine, amphetamine, MDMA, MDA, pseudoephedrine, phenylpropanolamine or MBDB in 14 cases.
    Keywords:  Amphetamine-type stimulants; Blood; GC/MS; NPS; Phenethylamines; Synthetic cathinones; Urine
    DOI:  https://doi.org/10.1016/j.forsciint.2025.112469
  34. Int J Mol Sci. 2025 Mar 29. pii: 3167. [Epub ahead of print]26(7):
    Archaeal Lipids: Extraction, Separation, and Identification via Natural Product Chemistry Perspective.
    Tuo Li, Youyi Luo, Changhong Liu, Xuan Lu, Baomin Feng.
      Archaeal lipids, defining a primordial life domain alongside Bacteria and Eukarya, are distinguished by their unique glycerol-1-phosphate backbone and ether-linked isoprenoid chains. Serving as critical geochemical biomarkers, archaeal lipids like glycerol dialkyl glycerol tetraethers (GDGTs) underpin paleoclimate proxies, while their phylum-specific distributions illuminate phylogenetic divergence. Despite the maturity of Mass Spectrometry-based quantitative biomarkers-predominantly those with established structures-becoming well-established in geochemical research, systematic investigation of archaeal lipids as natural products has notably lagged. This deficit manifests across three key dimensions: (1) Extraction methodology lacks universal protocols adapted to diverse archaeal taxa and sample matrices. While comparative studies exist, theoretical frameworks guiding method selection remain underexplored. (2) Purification challenges persist due to the unique structures and complex isomerization profiles of archaeal lipids, hindering standardized separation protocols. (3) Most critically, structural characterization predominantly depends on decades-old foundational studies. However, the existing reviews prioritize chemical structural, biosynthetic, and applied aspects of archaeal lipids over analytical workflows. This review addresses this gap by adopting a natural product chemistry perspective, integrating three key aspects: (1) the clarification of applicable objects, scopes, and methodological mechanisms of various extraction technologies for archaeal lipids, encompassing both cultured and environmental samples; (2) the elucidation of separation principles underlying polar-gradient lipid fractionation processes, leveraging advanced chromatographic technologies; (3) the detailed exploration of applications for NMR in resolving complex lipid structures, with specialized emphasis on determining the stereochemical configuration. By synthesizing six decades of methodological evolution, we establish a comprehensive analytical framework, from lipids extraction to structural identification. This integrated approach constructs a systematic methodological paradigm for archaeal lipid analysis, bridging theoretical principles with practical implementation.
    Keywords:  archaea; extraction; identification; lipids; natural product chemistry; separation
    DOI:  https://doi.org/10.3390/ijms26073167
  35. Analyst. 2025 Apr 17.
    Donor-acceptor covalent organic framework nanofilm-based laser desorption/ionization mass spectrometry for rapid and sensitive determination of creatinine in human serum.
    Hang Su, Zihan Chen, Juan Lin, Yanhui Zhong, Dan Ouyang, Zian Lin.
      Creatinine (Cre), a metabolite generated by muscles and kidneys, holds significant importance in clinical screening and detection of kidney disease. However, the existing clinical detection of Cre, such as the Jaffe reaction-based colorimetric method, requires complex sample pretreatment and is subject to interference in biological samples. Herein, a surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) method based on a donor-acceptor covalent organic framework (D-A COF) nanofilm as a substrate was proposed for Cre determination in human serum. The D-A COF nanofilm was synthesized using a solvothermal reaction on indium-tin-oxide (ITO)-coated glass plates, which featured uniform surfaces, good thermal stability, and excellent UV absorption. Compared with conventional organic matrices, the D-A COF nanofilm-based LDI-MS method showed low background interference and high MS response and was successfully used for the analysis of low-weight molecules, such as amino acids, bisphenols, and estrogens. On this basis, the D-A COF nanofilm-based LDI-MS method was developed for the determination of Cre in human serum. This method showed good linearity in the range of 14.0-750.0 μmol L-1 with a low limit of detection (LOD) of 4.5 μmol L-1, making it suitable for the determination of Cre in human serum with different concentration levels. This work demonstrates the potential of this method for the clinical determination of Cre in human serum and provides a new direction for the screening and determination of other small-molecule clinical markers.
    DOI:  https://doi.org/10.1039/d5an00317b
  36. Anal Chem. 2025 Apr 16.
    Comprehensive Metabolite Profiling in Single-Cell Systems via Dual-Modal MALDI-Mass Spectrometry Imaging.
    Jie Yuan, Xiafei Li, Xinxin Shen, Pei Xiong, Nanlin Zhu, Yang Ye, Jia Liu.
      The development of spatial multiomics technologies, particularly matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), has revolutionized our ability to map metabolic processes at single-cell resolution. However, the current techniques face challenges in minimizing matrix interferences and achieving comprehensive metabolite detection across multiple ionization modes. In this study, we present a novel dual-modal MSI workflow that leverages the pairing of 1,5-diaminonaphthalene (DAN) and its hydrochloric salt (DAN-HCl) matrices for sequential detection in positive- and negative-ion modes, respectively. This approach significantly enhanced metabolite coverage, spanning both lipid-based and nonlipid small molecules, while eliminating the need for solvent cleaning steps. Applied to a coculture of cholangiocarcinoma (CCLP1) and hepatic stellate (LX2) cells, the workflow revealed significant metabolic distinctions, including differential accumulation of glycerolipids and energy-related metabolites, highlighting the unique metabolic profiles of each cell type. Additionally, several unidentified metabolites were detected, indicating the potential to discover novel metabolic variations. These findings establish our method as a robust tool for single-cell spatial metabolomics with broad applicability in studying complex cellular interactions and advancing both research and clinical applications.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05480
  37. Food Chem. 2025 Apr 06. pii: S0308-8146(25)01482-7. [Epub ahead of print]483 144231
    MEATiCode: A comprehensive proteomic LC-MS/MS method for simultaneous species identification in meat authentication.
    Renata G Duft, Julian L Griffin, David A Stead.
      Food fraud in the meat industry threatens consumer trust, market stability, and public health. Traditional methods like DNA barcoding are limited, especially for processed foods where DNA is often degraded. This paper introduces the workflow MEATiCode, a comprehensive proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous identification of species in meat authentication. Application of a novel database search approach - MEATiCode - enabled the differentiation of meat species (as demonstrated for beef, pork, chicken and lamb) in raw and cooked food products following a simple sample preparation procedure and LC-MS/MS analysis of extracted meat peptides. The efficacy of the MEATiCode method was demonstrated through its application to a range of meat products, achieving high sensitivity (0.5 % Limit of Detection (LoD)) and reliability in the detection of adulteration, even in highly processed or cooked meats.
    Keywords:  Food fraud; Liquid chromatography mass spectrometry; Meat authentication; Proteomics
    DOI:  https://doi.org/10.1016/j.foodchem.2025.144231
  38. Foods. 2025 Mar 26. pii: 1147. [Epub ahead of print]14(7):
    Quantitative Analysis of Pyrrolizidine Alkaloids in Food Matrices and Plant-Derived Samples Using UHPLC-MS/MS.
    Runfeng Lin, Jing Peng, Yingjie Zhu, Suhe Dong, Xin Jiang, Danning Shen, Jiaxin Li, Peihong Zhu, Jie Mao, Na Wang, Kun He.
      Pyrrolizidine alkaloids (PAs) are a class of nitrogen-containing basic organic compounds that are frequently detected in foods and herbal medicines. Owing to their potential hepatotoxic, genotoxic, and carcinogenic properties, PAs have become a significant focus for monitoring global food safety. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the detection and analysis of three foods (tea, honey, and milk) susceptible to PA contamination. This optimized method effectively separated and detected three types of PAs, namely, three pairs of isomers and two pairs of chiral compounds. The limits of detection (LODs) and limits of quantification (LOQs) were determined to be 0.015-0.75 and 0.05-2.5 µg/kg, respectively, with the relative standard deviations (RSDs) of both the interday and intraday precisions remaining below 15%. The average PA recoveries from the honey, milk, and tea matrices fell within the ranges of 64.5-103.4, 65.2-112.2, and 67.6-107.6%, respectively. This method was also applied to 77 samples collected from 33 prefecture-level cities across 16 provinces and included 40 tea, 6 milk, 8 honey, 14 spice, and 9 herbal medicine samples. At least one PA was detected in twenty-three of the samples, with herbal medicines exhibiting the highest total PA content. The obtained results indicate that the developed method demonstrated good repeatability and stability in the detection and quantitative analyses of PAs in food- and plant-derived samples. This method is therefore expected to provide reliable technical support for food safety risk monitoring.
    Keywords:  UHPLC—MS/MS; food safety; honey; milk; pyrrolizidine alkaloids; tea
    DOI:  https://doi.org/10.3390/foods14071147
  39. bioRxiv. 2025 Apr 03. pii: 2025.04.02.646861. [Epub ahead of print]
    An LC-MS/MS method for the quantification of tobacco-specific carcinogen protein adducts.
    Breanne Freeman, Chengguo Xing.
      4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methylnitrosamino)-l-(3-pyri-dine)-l-butanol (NNAL) are tobacco-specific lung carcinogens. Methods have been developed to quantify NNK- and NNAL-specific DNA adducts in pre-clinical samples but are less feasible to translation due to limited access to target tissues for sufficient DNA. NNAL-specific DNA or protein adducts have never been detected in clinical samples, which are critical to support the physiological relevance of NNAL bioactivation and carcinogenesis. We reported a sensitive and specific LC-MS/MS method to quantify hydrolyzed NNAL adduct, 1-(3-pyridinyl)-1,4-butanediol (PBD). The method was applicable to variety of biological samples, to assess tobacco exposure and estimate NNAL bioactivation.
    DOI:  https://doi.org/10.1101/2025.04.02.646861
  40. Biomed Chromatogr. 2025 May;39(5): e70088
    Pharmacokinetics and Tissue Distribution Study for 2-(2-Fluorophenyl)-5-Phenyl-7-Alkoxy- [1,2,4]Triazole[1,5-a]Pyrimidine Antiepileptic Compounds in Rats.
    Han Liang, Ranran Guo, Yuxi Zhou, Cui Lv, Chuanlong Guo, Feng Su, Qingkun Dong, Longjiang Huang, Wen Xu.
      The development of novel active molecules with a clear target has always been an urgent need for the treatment of epilepsy. Previously, we reported 2-(2-fluorophenyl)-5-phenyl-7-propyl-[1,2,4]triazolo[1,5-a]pyrimidine (10C) as a selective and positive modulator of GABAA receptors, which exhibited excellent antiepileptic activity in mice with an ED50 value of 8.51 mg/kg. However, the pharmacokinetics (PK) profiles of this compound remain unclear. In this study, 10C and four analogs (10A, 10B, 10D, and 10E) as well as deuterated 10C were synthesized with high efficiency under optimized reaction conditions, and deuterated 10C was employed as an internal standard. The concentrations of the five compounds in rat plasma and of 10C in tissue homogenate were assayed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The results indicated that, compared to the other four analogs, 10C exhibited the highest drug concentration in rat plasma at the same dosage, which provides a good explanation for its superior antiepileptic activity compared to the other four analogs. Following intraperitoneal injection, 10C displayed favorable pharmacokinetic characteristics (F = 41%) and excellent brain penetration potency (B/P = 1.9). Overall, compound 10C is a promising lead for the research and development of new small-molecule therapeutics for epilepsy.
    Keywords:  epilepsy; liquid chromatography‐mass spectrometry; pharmacokinetics; tissue distribution
    DOI:  https://doi.org/10.1002/bmc.70088
  41. J Am Soc Mass Spectrom. 2025 Apr 18.
    Improving MALDI Mass Spectrometry Imaging Performance: Low-Temperature Thermal Evaporation for Controlled Matrix Deposition and Improved Image Quality.
    Toufik Mahamdi, Cristina Gomez Serna, Roger Giné, Jordi Rofes, Shad Arif Mohammed, Pere Ràfols, Xavier Correig, María García-Altares, Carsten Hopf, Stefania-Alexandra Iakab, Oscar Yanes.
      The deposition of matrix compounds significantly influences the effectiveness of matrix-assisted laser desorption/ionization (MALDI) Mass Spectrometry Imaging (MSI) experiments, impacting sensitivity, spatial resolution, and reproducibility. Dry deposition methods offer advantages by producing homogeneous matrix layers and minimizing analyte delocalization without the use of solvents. However, refining these techniques to precisely control matrix thickness, minimize heating temperatures, and ensure high-purity matrix layers is crucial for optimizing MALDI-MSI performance. Here, we present a novel approach utilizing low-temperature thermal evaporation (LTE) for organic matrix deposition under reduced vacuum pressure. Our method allows for reproducible control of matrix layer thickness, as demonstrated by linear calibration for two organic matrices, 2,5-dihydroxybenzoic acid (DHB) and 1,5-diaminonaphthalene (DAN). The environmental scanning electron microscopy images reveal a uniform distribution of small-sized matrix crystals, consistently on the sub-micrometer scale, across tissue slides following LTE deposition. Remarkably, LTE serves as an additional purification step for organic matrices, producing very pure layers irrespective of initial matrix purity. Furthermore, stability assessment of MALDI-MSI data from mouse brain sections coated with LTE-deposited DHB or DAN matrix indicates minimal impact on ionization efficiency, signal intensity, and image quality even after storage at -80 °C for 2 weeks, underscoring the robustness of LTE-deposited matrices for MSI applications. Comparative analysis with the spray-coating method highlights several advantages of LTE deposition, including enhanced ionization, reduced analyte diffusion, and improved MSI image quality.
    Keywords:  Dry Deposition; Low Temperature Thermal Evaporation; MALDI; Mass Spectrometry Imaging
    DOI:  https://doi.org/10.1021/jasms.5c00015
  42. RSC Adv. 2025 Apr 09. 15(15): 11478-11490
    Sensitive determination of chlorpromazine in pharmaceutical formulations and biological fluids using solid phase extraction followed by HPLC-UV and spectrophotometric analysis.
    Raed F Hassan, Jalal N Jeber, Hussein Fares Abd-Alrazack, Mohammad K Hammood, Muhannad M Abd.
      An environmentally friendly analytical method was developed to detect trace amounts of chlorpromazine in pharmaceutical formulations, urine, and serum samples. The method integrates magnetic solid-phase extraction (MSPE) with UV detection (MSPE-UV), utilizing environmentally friendly materials and procedures. The MSPE system employed 3-chloropropyltriethoxysilane-coated magnetic nanoparticles (Fe3O4@CPTES) as adsorbents, offering a sustainable and efficient alternative to conventional methods. The proposed technique eliminates the need for toxic organic solvents commonly used in pre-concentration steps, aligning with the principles of green chemistry. Optimization of extraction parameters, including pH, ionic strength, and adsorbent dosage, revealed high extraction efficiencies (98% for pharmaceutical solutions, 52% for urine, and 33% for serum), with corresponding enrichment factors of 102, 52, and 41, respectively, and detection limits as low as 0.08 ng mL-1 for pharmaceutical solutions. The method demonstrates excellent linearity (R 2 > 0.9988) over a wide range (0.15-400 ng mL-1) and high precision (RSD < 7%). The Fe3O4@CPTES nanoparticles enable rapid, reusable, and efficient extraction, reducing both analysis time and resource consumption. While tablet analysis validates method robustness, the primary application lies in trace CPZH detection in biological matrices. This green analytical approach offers a reliable, sensitive, and eco-friendly protocol for CPZH detection, highlighting its potential for broader applications in pharmaceutical and biomedical analysis.
    DOI:  https://doi.org/10.1039/d5ra01298h
  43. Food Chem Toxicol. 2025 Apr 11. pii: S0278-6915(25)00215-7. [Epub ahead of print]201 115447
    GC-MS and HPLC-FLD for sensitive assay of toxic cyclohexylamine in artificial sweetener tablets and human biological fluids.
    Hazim M Ali, Amr A Essawy, Ahmed Hamad Alanazi, Arafa Musa, Ahmed Emad F Abbas, Ibrahim Bayoumi Abdel-Farid, Mohammed Gamal.
      Cyclohexylamine (CHA) is a precursor in the synthesis of artificial sweeteners cyclamate and its major metabolite. CHA is toxic to the nervous system, kidneys, and liver in animal studies. In the present work, gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-fluorescence detection (HPLC-FLD) were introduced for the first time to the determination of nano-level concentration of CHA in artificial sweetener tablets and human biological fluids. According to the obtained results, HPLC-FLD and GC-MS of CHA exhibit a wide linear range from 1 to 1250 ng/mL and 5-1500 ng/mL within limits of detection of 0.49 and 1.55 ng/mL, respectively. Furthermore, both chromatographic techniques exhibited high accuracy and precision, yielding recovery estimates ranging from 95.85 % to 100.70 % for the HPLC-FLD method and 91.54 %-99.64 % for the other while the corresponding values of relative standard deviation (RSD%) range from 0.25 to 0.82 % and 1.25-3.97 %. The accuracies for HPLC-FLD and GC-MS method in serum and urine samples are within the range of 90.29-99.94 %. On the other hand, CHA was detected in all studied artificial sweetener tablets, its level ranged from 1.35 to 102.64 ng/tablet. Also, the results obtained from the HPLC-FLD and GC-MS methods were statistically compared, and no significant difference was found.
    Keywords:  Artificial sweetener tablets; Cyclamate; Cyclohexylamine; GC-MS; HPLC-FLD
    DOI:  https://doi.org/10.1016/j.fct.2025.115447
  44. Sci Rep. 2025 Apr 16. 15(1): 13155
    Human plasma protein bindings of neonicotinoid insecticides and metabolites.
    Kumiko Taira, Yoshinori Ikenaka, Jean-Marc Bonmatin, Anton Safer.
      Neonicotinoid insecticides (neonicotinoids) are widely used in agriculture, forestry and public health in the world. Environmental exposure to neonicotinoids has been increasing due to their continuous uses. Neonicotinoids act as agonists, antagonists, or modulators of acetylcholine receptors and have adverse effects on non-target species, such as invertebrates, amphibians, reptiles, birds, microbes and mammals. Although there is concern about their adverse effects on ecosystem services and their potential effects on human health, their xenobiotic kinetics and dynamics in humans are not understood well. In this study, we determined a xenobiotic kinetic parameter, plasma protein bindings (PPBs) of 7 neonicotinoids and 18 metabolites with human plasma using a Rapid Equilibrium Dialysis (RED) device and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and compared their PPBs with their physicochemical properties. 6-chloronicotinic acid (6-CNA) exhibited the highest PPB (86.4%), followed by imidacloprid-olefin (86.3%) in human plasma. Their PPBs are much higher than that of the parent compound, imidacloprid (27.5%). The PPBs of neonicotinoids and metabolites are not related to their lipophilicity determined by reversed-phase LC. The results shed light on the behavior of environmentally exposed neonicotinoids and metabolites and warrant further research on their xenobiotic kinetics and dynamics in humans.
    DOI:  https://doi.org/10.1038/s41598-025-96812-y
  45. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Apr 11. pii: S1570-0232(25)00144-8. [Epub ahead of print]1258 124592
    Recent advancements in the bioactive alkaloids analysis in plant and biological specimen: From the perspective of activity, sample preparation, and analytical method selection.
    Sevilay Erdogan-Kablan, Seyda Yayla, M Mesud Hurkul, Ahmet Cetinkaya, Emirhan Nemutlu, Sibel A Ozkan.
      Alkaloids are a diverse group of naturally occurring organic compounds. They are known for their significant pharmacological properties. This review provides an up-to-date analysis of bioactive alkaloids in plant and biological samples, emphasizing their biological activities, extraction techniques, and analytical methods. The study focuses on significant alkaloids such as morphine, codeine, vinblastine, vincristine, berberine, quinine, quinidine, caffeine, nicotine, ephedrine, and atropine, highlighting their chemical structures, therapeutic applications, and mechanisms of action. Recent advances in extraction methods, including conventional and modern green techniques such as supercritical fluid extraction, microwave-assisted extraction, and solid-phase microextraction, are discussed in detail. In addition, the review provides an overview of state-of-the-art analytical techniques used for alkaloid quantification, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, and novel spectroscopic methods. Emphasis is placed on the challenges associated with alkaloid analysis, including matrix effects, stability, and structural diversity. The results contribute to the growing body of knowledge in alkaloid research, providing insights into their potential therapeutic applications and analytical improvements for more accurate and efficient detection in various biological and plant matrices.
    Keywords:  Analytical methods; Biological activities; Plant alkaloids; Sample preparation
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124592
  46. Anal Chem. 2025 Apr 15.
    Rapid Screening of Illicit Drugs from Biofluid via Dried Blood/Urine Spot and Ultrasonic Desorption-Assisted Low-Temperature Arc Plasma Ionization Mass Spectrometry.
    Zhongbao Han, Zhongyu Zhao, Meiyun Pan, Yimeng Qi, Liyan Liu, Dan Wang, Zhan Yu.
      A novel method for rapid and sensitive illicit drug screening in biofluids has been developed by employing a paper-based sample collection coupled with ultrasonic desorption-assisted low-temperature plasma ionization mass spectrometry (PBS-LTPI-MS). For optimization, key experimental parameters, such as geometry coordinates, angle, and plasma intensity, were fine-tuned using ketamine as the representative analyte. The redissolution process, which encompasses the use of organic solvents and sample collection paper, underwent an evaluation to establish conditions conducive to efficient ionization. The proposed method demonstrated excellent analytical performance, with linear ranges exceeding R2 > 0.99, limits of detection ranging from 10 to 20 ng mL-1, and impressive recovery rates exceeding 91.16% in complex biofluid matrixes. Spiked recovery experiments revealed strong matrix tolerance and reliable performance, even in the presence of illicit drug mixtures. The robustness of the sampling device under varying storage temperatures and durations further confirmed the method's suitability for point-of-care testing and large-scale sample collection. With the ability to analyze samples within a mere 3 s, high-throughput potential, and environmental robustness, this method stands out as an invaluable instrument for rapid illicit drug screening and forensic analysis.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00232
  47. Comput Methods Biomech Biomed Engin. 2025 Apr 15. 1-13
    A deep learning framework for enhanced mass spectrometry data analysis and biomarker screening.
    Shuyu Zhang, Zhiyu Li, Weili Peng, Yuanyuan Chen, Yao Wu.
      Mass spectrometry (MS) serves as a powerful analytical technique in metabolomics. Traditional MS analysis workflows are heavily reliant on operator experience and are prone to be influenced by complex, high-dimensional MS data. This study introduces a deep learning framework designed to enhance the classification of complex MS data and facilitate biomarker screening. The proposed framework integrates preprocessing, classification, and biomarker selection, addressing challenges in high-dimensional MS analysis. Experimental results demonstrate significant improvements in classification tasks compared to other machine learning approaches. Additionally, the proposed peak-preprocessing module is validated for its potential in biomarker screening, identifying potential biomarkers from high-dimensional data.
    Keywords:  Liver disease; deep learning; mass spectrometry; pre-processing
    DOI:  https://doi.org/10.1080/10255842.2025.2488501
  48. Forensic Toxicol. 2025 Apr 14.
    Preparation of highly concentrated extracts from large volume of urine as the first step in detecting trace amounts of hypnotics in urine collected in drug-facilitated crime cases.
    Kenji Kuwayama, Hajime Miyaguchi, Tatsuyuki Kanamori, Kenji Tsujikawa, Tadashi Yamamuro, Hiroki Segawa, Yuki Okada, Yuko T Iwata.
       PURPOSE: Detecting hypnotics in victim urine samples collected several days after drug-facilitated crime (DFC) is challenging because most of the drugs have already been excreted. In this study, a sample preparation method was developed for extracting trace amounts of hypnotics using most of the urine excreted at one sampling time (100 mL), and large amounts of matrices were efficiently removed.
    METHODS: Etizolam, midazolam, ramelteon, and their metabolites were used as the target compounds. As the first step in decreasing the sample volume, solid-phase extraction using various sorbents was examined. The effects of additional clean-up columns (alumina, graphite, anion exchanger, etc.) on the removal of urine matrices were also examined. The pretreatment of 0.1-mL urine using a simple extraction column, specialized for small-scale urinalysis (Isolute Hydro DME +), was used as the reference method. The feasibility of drug detection in 100-mL urine was evaluated by comparison with a reference method.
    RESULTS: All analytes in 100-mL urine were most effectively adsorbed on a sorbent with octadecyl-bonded polymer and eluted with less than 2 mL of acetonitrile. A multilayer clean-up column consisting of alumina, octadecyl-bonded silica, and anion exchangers was effective in removing the matrices. α-Hydroxymidazolam was detected in 100 mL of urine that was collected 5 days after midazolam administration, but was undetected using the reference method.
    CONCLUSIONS: This preparation method for 100-mL urine is useful as the first extraction step in detecting trace amounts of hypnotics in victim urine collected late after DFC.
    Keywords:  Drug-facilitated crime; Hypnotics; Liquid chromatography/mass spectrometry; Solid-phase extraction; Urine
    DOI:  https://doi.org/10.1007/s11419-025-00722-7
  49. J Am Soc Mass Spectrom. 2025 Apr 18.
    Fourier Transform Data Analysis for Mass Spectra of Small Polymers and Pyrolysates with Accurate-Mass Data and Mass Defect Preprocessing.
    Robert B Cody.
      Early applications of Fourier transform (FT) data analysis of polymer mass spectra made use of nominal-mass spectra to determine the monomer mass. More recently, FT data analysis has been applied to the interpretation of the unresolved envelope in complex electrospray ionization mass spectra of ionic polymers and macromolecules. Here, FT data analysis is applied to determine the monomer composition for isotopically resolved accurate-mass MALDI, pyrolysis DART, and PaperSpray mass spectra of synthetic homopolymers, copolymers, and polymer mixtures. Monomer compositions are determined from the transformed data by an accurate mass search and elemental composition calculations. Data analysis is facilitated for complex mass spectra containing multiple polymers, multiple charge states, and nonperiodic fragments by graphically selecting individual series from mass defect plots.
    DOI:  https://doi.org/10.1021/jasms.5c00030
  50. J Pharm Biomed Anal. 2025 Apr 08. pii: S0731-7085(25)00223-7. [Epub ahead of print]262 116882
    Unique methotrexate polyglutamates distributions in peripheral blood mononuclear cells of rheumatoid arthritis patients: Development and validation of a UPLC-MS/MS method.
    Janani Sundaresan, Marry Lin, Gerrit Jansen, Renske C F Hebing, Maja Bulatović-Ćalasan, Robert de Jonge, Eduard A Struys, Maurits C F J de Rotte.
      Methotrexate is pivotal in treating immune-mediated inflammatory diseases. Intracellularly, methotrexate is metabolized to methotrexate-polyglutamates (MTX-PG1-7), comprising up to six additional glutamate moieties, crucial for cellular retention and therapeutic efficacy. Hitherto, quantification of MTX-PG1-6 in peripheral blood mononuclear cells (PBMCs) from methotrexate-treated patients was challenging due to their low abundance in blood and matrix effects. We present a robust validated UPLC-MS/MS method to quantify individual MTX-PG1-6 in PBMCs. Stable-isotope labelled internal standard mixture of MTX-PG1-6 was added to 5 million PBMCs, followed by deproteinization with perchloric acid, and additional sample clean-up using solid phase extraction columns. MTX-PG1-6 were detected and quantified using UPLC-MS/MS. The method was validated for lower limit of quantification (LLOQ), linearity, carryover, recovery, matrix effects, precision and stability. We assessed MTX-PG1-6 in PBMCs derived from five methotrexate-treated rheumatoid arthritis patients. For all MTX-PG1-6, LLOQs were < 1 fmol-MTX-PG1-6/million cells with linearities R2 > 0.995. The recoveries, carryover and stability were acceptable and no matrix effects were observed. The intraday and interday precision %CVs of quality controls ranged from 2.7 % to 11.4 % and 3.5-14.9 % respectively. Interday precision using nine PBMCs aliquots from a single MTX-treated patient aligned similarly (%CV <15 %). In patient-derived PBMC samples, MTX-PG1 was the highest, with decreasing concentrations of MTX-PG2 to MTX-PG5. No signal for MTX-PG6 was detected in the patient samples. We validated a new UPLC-MS/MS method to quantify MTX-PG1-6 in PBMCs, thus facilitating PBMC-based therapeutic drug monitoring studies and understand associations between MTX-PG1-6 concentration and therapy efficacy or adherence.
    Keywords:  Immune mediated inflammatory diseases; Methotrexate polyglutamates in PBMCs; Therapeutic drug monitoring
    DOI:  https://doi.org/10.1016/j.jpba.2025.116882
  51. Food Chem. 2025 Apr 06. pii: S0308-8146(25)01462-1. [Epub ahead of print]483 144211
    Comprehensive review of pyrrolizidine alkaloids in bee products: Occurrence, extraction, and analytical methods.
    Patricia Brugnerotto, Bibiana Silva, Luciano Valdemiro Gonzaga, Ana Carolina Oliveira Costa.
      Pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) are hepatotoxic secondary metabolites present in certain plant genera, raising health concerns due to their inevitable occurrence in bee products like honey, pollen, royal jelly, and propolis. The European Commission has set a 500 μg kg-1 limit for PAs/PANOs in pollen-based supplements to ensure safety, emphasizing the need for sensitive analytical methods. This review, based on studies published between 2019 and 2024, identifies 51 compounds in bee products, including 32 PAs and 19 PANOs, with lycopsamine, senecionine, echimidine, intermedine, and retrorsine being the most studied. Solvent extraction, often combined with SPE or QuEChERS, is the most used preparation method, while liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is preferred for detection. Few studies assessed the risk of PAs consumption. These findings support regulatory monitoring of bee product safety and highlight the need for refining detection methods and establishing standardized limits and monitoring for PAs/PANOs.
    Keywords:  Honey; Liquid chromatography-tandem mass spectrometry; Pollen; QuEChERS; Solid-phase extraction; Solvent extraction; Toxicity
    DOI:  https://doi.org/10.1016/j.foodchem.2025.144211
  52. Doc Ophthalmol. 2025 Apr 12.
    The ERGtools2 package: a toolset for processing and analysing visual electrophysiology data.
    Moritz Lindner.
       PURPOSE: To introduce ERGtools2, an open-source R package for processing, analysing and long-term storing visual electrophysiology data.
    METHODS: A dataset comprising Electroretinogram (ERG) recordings of C57Bl/6J mice, subjected to standard ISCEV stimuli, was used to present the functionality of ERGtools2. ERGtools2 stores and organizes all recordings, metadata, and measurement information from an individual examination in a single object, maintaining raw data throughout the analysis process.
    RESULTS: A standard workflow is presented exemplifying how ERGtools2 can be used to efficiently import, pre-process and analyse ERG data. Following this workflow, basic ERG measurements and visualisation of a single exam as well as group statistics are obtained. Moreover, special use cases are described, including for the handling of noisy data and the storage of data in the HDF5 format to ensure long-term preservation and accessibility.
    CONCLUSIONS: ERGtools2 provides a comprehensive, flexible, and device-independent solution for visual electrophysiology data analysis. Its emphasis on maintaining raw data integrity, combined with advanced processing and analysis capabilities, makes it a useful tool for preclinical and clinical research applications. The open-source nature and the use of open data formats promote reproducibility and data sharing in visual neurosciences.
    Keywords:  Data analysis; Data preservation; Open source; R package; Visual electrophysiology
    DOI:  https://doi.org/10.1007/s10633-025-10017-2
  53. J Nutr. 2025 Apr 16. pii: S0022-3166(25)00227-5. [Epub ahead of print]
    Endogenous metabolites in metabolic diseases: pathophysiological roles and therapeutic implications.
    Mengjie Xiao, Ning Zhou, Zhen Tian, Changhao Sun.
      Breakthroughs in metabolomics technology have revealed the direct regulatory role of metabolites in physiology and disease. Recent data have highlighted the bioactive metabolites involved in the etiology and prevention, and treatment of metabolic diseases such as obesity, nonalcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus (T2DM), and atherosclerosis. Numerous studies reveal that endogenous metabolites biosynthesized by host organisms or gut microflora regulate metabolic responses and disorders. Lipids, amino acids, and bile acids (BAs), as endogenous metabolic modulators, regulate energy metabolism, insulin sensitivity, and immune response through multiple pathways, such as insulin signaling cascade, chemical modifications, and metabolite-macromolecule interactions. Furthermore, the gut microbial metabolites short-chain fatty acids (SCFAs), as signaling regulators have a variety of beneficial impacts in regulating energy metabolic homeostasis. In this review, we will summarize information about the roles of bioactive metabolites in the pathogenesis of many metabolic diseases. Furthermore, we discuss the potential value of metabolites in the promising preventive and therapeutic perspectives of human metabolic diseases.
    Keywords:  gut microbiome; metabolic diseases; metabolites; prevention and therapy
    DOI:  https://doi.org/10.1016/j.tjnut.2025.04.017
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