bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–06–15
fifty-two papers selected by
Sofia Costa, Matterworks
  1. Nontargeted Metabolomics Using the Sciex ZenoTOF 7600.
  2. Comparison of Untargeted LC-MS/MS Metabolomics Data Processing Through Compound Discoverer and MS-DIAL.
  3. Revealing the Metabolome: Metabolite Workflow for Retinal Ganglion Cells.
  4. Chromatographic Choice in Cerebrospinal Fluid Metabolomics Identification Outcome : Metabolomics for Cerebrospinal Fluid.
  5. Simultaneous determination of multiple neurochemicals in mouse brain tissue by liquid chromatography-tandem mass spectrometry.
  6. Untargeted Metabolomic Analysis in the Short-Lived Killifish Using HILIC Ultra Performance Liquid Chromatography Method Coupled to a Q Exactive Orbitrap Mass Spectrometer.
  7. Quantitation of Tuvusertib (M1774) in Human Plasma by LC-MS/MS.
  8. Development and Validation of a Simple and Cost-Effective LC-MS/MS Method for the Quantitation of the Gut-Derived Metabolite Trimethylamine N-Oxide in Human Plasma of Healthy and Hyperlipidemic Volunteers.
  9. Isotopic Ratio Outlier Analysis (IROA) for Quantitative Analysis.
  10. Quantification of galacturonic acid in pectin-containing samples using a stable isotope dilution approach and LC-MS.
  11. Automatic Magnetic Dispersive Solid-Phase Extraction Combined With LC-MS/MS for the Rapid Quantitation of Six Gelsemium elegans Alkaloids in Biological Samples.
  12. Polyamine Metabolism in Optic Nerve: Spatial Metabolomics with AP/MALDI Mass Spectrometry Imaging.
  13. LC-MS/MS-based simultaneous quantification of acylcarnitines, eicosapentaenoic acid, and docosahexaenoic acid: exploring potential biomarkers in human plasma.
  14. Quantification of Melatonin with Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry.
  15. A sex-sensitive LC-MS/MS method with isotopic internal standard for prazosin bioequivalence: bridging precision medicine and generic drug policy in China.
  16. Reproducible Sectioning and Sample Preparation for Mass Spectrometry Imaging of Cancer Metabolism in Frozen Lung Tissue.
  17. Spatial Atmospheric Pressure MALDI HRMS Analysis of Histological Sections of FFPE Samples Reveals a Unique Subset of the Metabolome.
  18. NMR Metabolomics in Livestock: An Update of Current Methods and Applications.
  19. Analysis of Soybean Phospholipid Metabolites Using Triple Quadrupole Mass Spectrometry.
  20. DANGO: An MS data annotation tool for glycolipidomics.
  21. LC-MS-Based Metabolic Fingerprinting of Vitreous Humor.
  22. Enrichment analysis for spatial and single-cell metabolomics accounting for molecular ambiguity.
  23. Direct Colony Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Assay for Targeted Operon Metabolite Analysis.
  24. Development and validation of a high-throughput LC-MS/MS bioanalytical method for the simultaneous quantification of cannabidiol and metabolites in human plasma.
  25. Untargeted Metabolomics of Human Embryonic Kidney (HEK) Cell Line.
  26. An Innovative Strategy for Untargeted Mass Spectrometry Data Analysis: Rapid Chemical Profiling of the Medicinal Plant Terminalia chebula Using Ultra-High-Performance Liquid Chromatography Coupled with Q/TOF Mass Spectrometry-Key Ion Diagnostics-Neutral Loss Filtering.
  27. Development of a quantitative structure-response relationships to estimate concentrations of plasticizer metabolites in urine without reference standards using non-targeted analysis with liquid chromatography high-resolution mass spectrometry.
  28. Advances in metallic nanostructures-assisted laser desorption/ionization mass spectrometry imaging of biological samples: A review.
  29. Neural Spectral Prediction for Structure Elucidation with Tandem Mass Spectrometry.
  30. The microbiome diversifies long- to short-chain fatty acid-derived N-acyl lipids.
  31. Multi-residue environmental method for seed treatment insecticides, neonicotinoid degradation products, and fungicides using liquid chromatography tandem mass spectrometry with a new ionization source.
  32. High-spatial-resolution mass spectrometry imaging of biological tissues using a microfluidic probe.
  33. A New, Fast, and Sensitive UPLC-ESI-MS/MS Method for the Simultaneous Analysis of 13 Neurotransmitters in Rat Brain.
  34. Utilizing the total phosphorus content of lipid extracts as a normalization strategy in quantitative lipidomics studies.
  35. Quantitative Chiral Separations on KRAS Inhibitor Sotorasib by UHPLC-MS/MS: Method Validation and Enantioselective Pharmacokinetics Assessment in Rat Plasma.
  36. A fully automated, high-throughput electro-extraction and analysis workflow for acylcarnitines in human plasma and mouse muscle tissues.
  37. Cytokine profiling in COVID-19 patients by using ion mobility-mass spectrometry-based parallel reaction monitoring.
  38. Metabolomics of the Kynurenine Pathway by Laser Desorption Ionization Mass Spectrometry (LDI-MS).
  39. Controllable Ion-Repulsion Enables Rapid and Trace-Level Detection of Hydrophobic Sulfonylurea Herbicides with ICPMS/MS without the Organic Mode.
  40. Simultaneous determination of 12 fluorescent whitening agents in paper and plastic food packaging materials for infants by high-performance liquid chromatography-tandem mass spectrometry.
  41. Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD + metabolites in CD38 knockout mice exhibiting improved metabolism.
  42. Bioanalytical Method Development and Validation of Dapagliflozin in Human Plasma Using RP-HPLC Method.
  43. High-Temperature Liquid Chromatography of Isomeric Organochlorine Herbicides and Their Degradation Products.
  44. An LC/MS/MS method for quantifying 25-hydroxyvitamin D3 in finger-prick plasma sample prepared by DEMECAL® micro plasma separation device: toward mail-in assessment of vitamin D status.
  45. Targeted Quantitation of 6ppd-Quinone in Fish Tissue Samples with Liquid Chromatography-Tandem Mass Spectrometry.
  46. Expanding the MR1 ligandome using chemical class-specific fragmentation and molecular networking.
  47. Separation and Quantification of Isomeric Forms of Aminobutyric Acids.
  48. Single-Cell Multiomic MALDI-MSI Analysis of Lipids and N-Glycans through Affinity Array Capture.
  49. Development of the High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Creatine, L-Carnitine, and Creatinine in Tears.
  50. Functionalized toothpicks for solid-phase microextraction of drugs from oral fluids.
  51. On Selecting Robust Approaches for Learning Predictive Biomarkers in Metabolomics Data Sets.
  52. Protocols for Metabolomic Analyses to Assess Changes in Central Carbon Metabolism from Neural Tissue.
  1. Methods Mol Biol. 2025 ;2925 1-23
    Nontargeted Metabolomics Using the Sciex ZenoTOF 7600.
    Anna Artati, Pauline Couacault, Michael Witting.
      Nontargeted metabolomics aims to capture as many metabolites as possible present in a biological system under a certain condition. Despite several analytical techniques commonly applied for nontargeted metabolomics, liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) is one of the most employed techniques. To cover broad metabolic classes, different chromatographic separation techniques are implemented, i.e., reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) techniques. Each method offers a complementary polarity range. While RP covers semi- to nonpolar metabolites, such as fatty acids, acylcarnitines, steroids, bile acids, etc., HILIC allows the analysis of polar metabolites, such as amino acids, amines, and organic acids.Nontargeted metabolomics platforms at Helmholtz Munich have been validated for various biological matrices, including biological liquids (plasma, serum, urine), tissues (liver, heart), and cell samples of human and rodent. Analysis is based on the Sciex ZenoTOF 7600 and three chromatographic separation methods, which can be used individually or in combination.
    Keywords:  Hydrophilic interaction liquid chromatography (HILIC); LC-HRMS/MS; Metabolomics; Non-targeted; Reversed phase (RP)
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_1
  2. Methods Mol Biol. 2025 ;2925 113-120
    Comparison of Untargeted LC-MS/MS Metabolomics Data Processing Through Compound Discoverer and MS-DIAL.
    Michael Coronado, Anh Pham, Sanjoy K Bhattacharya.
      Untargeted metabolomics is a technique used to detect the presence of all metabolites, or small-molecular-weight molecules, within a given sample. This technique is often accomplished through the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). LC-MS/MS provides raw data that require the use of bioinformatics software to extract significant features. Many bioinformatics software options are available, such as Compound Discoverer and MS-DIAL. This protocol details the processing of raw data through both software for functional comparison and optimization.
    Keywords:  Compound discoverer; Liquid chromatography; MS-DIAL; Mass spectrometry; Metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_7
  3. Methods Mol Biol. 2025 ;2925 271-288
    Revealing the Metabolome: Metabolite Workflow for Retinal Ganglion Cells.
    Alejandra Hernandez, Sofia Yarosh, Amr Almobayed, Sanjoy K Bhattacharya.
      In this chapter, we outline the preparation and isolation of retinal ganglion cells (RGCs) from postnatal mice. This includes a metabolite extraction method using 1:1 methanol/water from 6 or 12-well plate dishes. We provide liquid chromatography-mass spectrometry (LC-MS) parameters using a QExactive High Resolution Orbitrap Mass Spectrometer and HILIC column. The software used for analysis includes Compound Discoverer and MetaboAnalyst. Through this metabolic workflow, this protocol provides a biomarker discovery, allowing for future experimentation to treat and cure ocular neuropathies.
    Keywords:  Axon regeneration; Compound discoverer; Extraction; Mass spectrometry; MetaboAnalyst; Metabolomic profiling; Metabolomics; Retinal ganglion cells
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_20
  4. Methods Mol Biol. 2025 ;2925 91-101
    Chromatographic Choice in Cerebrospinal Fluid Metabolomics Identification Outcome : Metabolomics for Cerebrospinal Fluid.
    Sofía C Ayala Rodríguez, Alyssa M Suarez, Victor M Lu, Sanjoy K Bhattacharya, Ashish H Shah.
      Limited literature on the optimal chromatographic method for cerebrospinal fluid (CSF) extraction and identification is available. Ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) is an extensively used tool in metabolic profiling because of its high resolving power, analysis sensitivity, and selectivity. This technique allows for reliable structural elucidation for metabolite identification and quantification at low micromolar to nanomolar concentration levels. In this book chapter, we discuss two complementary UPLC-MS methods, reversed-phase (RP) and chromatography hydrophilic interaction liquid chromatography (HILIC), to characterize metabolomic analysis of CSF samples.
    Keywords:  Cerebrospinal fluid; Hydrophilic interaction liquid chromatography; Metabolomics; Reversed-phase C18; Ultraperformance liquid chromatography-mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_5
  5. Talanta. 2025 Jun 01. pii: S0039-9140(25)00911-7. [Epub ahead of print]296 128421
    Simultaneous determination of multiple neurochemicals in mouse brain tissue by liquid chromatography-tandem mass spectrometry.
    Yunji Kim, Jangjae Lee, Da Yong Lee, Subin Park, Jeongkwon Kim, Sung-Hee Cho.
      Neurochemicals play critical roles in modulating neuronal communication and brain functions, with their dysregulation resulting in neuropsychiatric disorders. The accurate quantification of these signaling molecules is essential for understanding disease mechanisms and developing therapeutics. To address the numerous drawbacks of current neurochemical analysis techniques, we herein developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 55 neurochemicals in biological samples. This method employs protein precipitation and a fluorophenyl column for chromatographic separation and does not require derivatization. The mobile phase, organic solvent, and column oven temperature were optimized to enhance sensitivity and selectivity. The validated method exhibited an excellent linearity (r2 > 0.990), limits of detection ranging from 0.15 to 75 ng/mL, limits of quantitation ranging from 0.5 to 250 ng/mL, and AOAC guideline-compliant intra- and interday precisions and accuracies. Using this method, we achieved separation within 13 min and quantified 45 neurochemicals in different mouse brain regions, revealing region-specific distributions. The developed approach holds promise for neuroscience research, clinical diagnostics, and the development of drug for neuropsychiatric disorder treatment.
    Keywords:  Fluorophenyl column; LC-MS/MS; Neurochemical; Simultaneous quantification
    DOI:  https://doi.org/10.1016/j.talanta.2025.128421
  6. Methods Mol Biol. 2025 ;2925 397-405
    Untargeted Metabolomic Analysis in the Short-Lived Killifish Using HILIC Ultra Performance Liquid Chromatography Method Coupled to a Q Exactive Orbitrap Mass Spectrometer.
    Isabella G Moceri, Selene Cabrera, Jackson Watson, Pieter-Jan Serneels, Julie De Schutter, Lieve Moons, Sanjoy K Bhattacharya.
      Over the past decade, advancements in liquid chromatography-mass spectrometry (LC-MS) have transformed untargeted metabolomic analysis. These improvements have led to biomarker discoveries, enabling scientists to deepen their understanding of biological systems in disease contexts. Achieving reproducibility, robustness, and reliable quantification is essential for confident metabolite identification. In this chapter, we present an untargeted metabolomic approach to analyze metabolites in the optic nerve tissue of adult Nothobranchius furzeri, an established biogerontology model that is increasingly used in tissue repair studies. The analysis is conducted using HILIC chromatography coupled with a Q Exactive Orbitrap mass spectrometer.
    Keywords:  HILIC; Killifish; Q Exactive Orbitrap; Ultra-performance liquid chromatography-mass spectrometry; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_26
  7. Biomed Chromatogr. 2025 Jul;39(7): e70134
    Quantitation of Tuvusertib (M1774) in Human Plasma by LC-MS/MS.
    Tien V Le, Robert A Parise, Julianne L Holleran, Christopher J Bakkenist, Timothy Synold, Ye Feng, Gregory M Cote, Steven D Gore, Jan H Beumer.
      Ataxia-telangiectasia and Rad3-related (ATR) protein kinase is an essential regulator of the DNA damage response (DDR) at stalled and collapsed replication forks. Tuvusertib (M1774) is a selective, orally available small molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. This study presents a robust and simple 5-min assay designed for the quantification of single agent tuvusertib in human plasma utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS). A 20 μL volume of plasma was subjected to protein precipitation, followed by chromatographic separation using a Phenomenex Synergi Polar-RP (4 μm, 2.1 × 50 mm) and a gradient mobile phase system consisting of 0.1% formic acid in both water and acetonitrile during a 4-min run time. Mass spectrometric detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, our assay met the criteria outlined by the Food and Drug Administration guidance for bioanalytical method validation, demonstrating robust performance within the range from 5 to 5000 ng/mL. This assay will support ongoing and future clinical studies by defining tuvusertib pharmacokinetics.
    Keywords:  LC‐MS/MS; M1774; human plasma; pharmacokinetics; tuvusertib; validation
    DOI:  https://doi.org/10.1002/bmc.70134
  8. Molecules. 2025 May 30. pii: 2398. [Epub ahead of print]30(11):
    Development and Validation of a Simple and Cost-Effective LC-MS/MS Method for the Quantitation of the Gut-Derived Metabolite Trimethylamine N-Oxide in Human Plasma of Healthy and Hyperlipidemic Volunteers.
    Nikolaos A Parisis, Panoraia Bousdouni, Aikaterini Kandyliari, Maria-Helen Spyridaki, Amalia Despoina Koutsogianni, Christina Telli, Konstantinos K Tsilidis, Antonios E Koutelidakis, Andreas G Tzakos.
      Trimethylamine N-oxide (TMAO) is a gut microbial metabolite of dietary precursors, including choline and carnitine. Elevated levels of TMAO in human plasma have been associated with several diseases such as cardiovascular, diabetes mellitus, chronic kidney disease, neurological disorders, and cancer. This has led to an increased interest in the accurate determination of TMAO in human blood, for which a reliable, cost-effective and sensitive analytical method should be established. LC-MS/MS has emerged as a powerful tool for the determination of TMAO due to its high sensitivity, specificity, and ability to handle complex matrices. Herein, we describe the development and validation of an LC-MS/MS method for the determination of TMAO in human blood plasma. Our method involves a simple sample preparation protocol, involving a protein precipitation step along with a non-deuterated IS, followed by a Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis using a triple quadrupole mass spectrometer. Additionally, the method was adapted and implemented on an UPLC-QTOF/MS. The method was validated using the guidelines set by the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for assay performance and robustness in human plasma and successfully applied to plasma derived from healthy and hyperlipidemic volunteers. The developed method was found to be specific, sensitive, and accurate for the determination of TMAO in human plasma, with a lower limit of quantification of 0.25 µM. The intra- and inter-assay precision and trueness were within acceptable limits.
    Keywords:  Rhodamine B; TMAO; biomarkers; human plasma; liquid chromatography; method validation; simple extraction methodology; tandem mass spectrometry
    DOI:  https://doi.org/10.3390/molecules30112398
  9. Methods Mol Biol. 2025 ;2925 69-89
    Isotopic Ratio Outlier Analysis (IROA) for Quantitative Analysis.
    Chris Beecher, Felice A de Jong.
      Metabolomics is an omics science that tries to measure the metabolic composition of a sample rapidly, but the current state of the art cannot provide the reproducibility or accuracy needed for clinical use or even daily reproducibility for larger experiments. The IROA TruQuant Workflow uses a daily long-term reference standard (LTRS) and a chemically identical Internal Standard (IS) to provide validated chemical identity, accurate reproducible quantitation, and daily QA/QC (Quality Assurance / Quality Control) on instrument and sample preparation. It does this for hundreds of compounds, generating comparable measurements across days, instruments, and chromatographic methods. All the compounds in the LTRS are isotopically signed with formula-indicating IROA patterns, ensuring they cannot be mistaken as artifacts. These patterns allow software-driven analysis to determine daily instrument performance in terms of sensitivity, in-source fragmentation, chromatographic and injection stability, and to provide reproducible quantitation.
    Keywords:  Clinical metabolomics; Dual-MSTUS Normalization; Error-corrected quantitation; IROA TruQuant Workflow; Isotopic ratio outlier analysis; MS error correction; Metabolic profiling; Metabolomics Internal Standard; Metabolomics normalization; Suppression correction
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_4
  10. Carbohydr Polym. 2025 Oct 01. pii: S0144-8617(25)00599-5. [Epub ahead of print]365 123816
    Quantification of galacturonic acid in pectin-containing samples using a stable isotope dilution approach and LC-MS.
    Johanna Braun, Mirko Bunzel.
      Reliable quantification of galacturonic acids (GalA) is essential for understanding the structural and functional properties of pectin-containing materials. However, conventional photometric methods often suffer from low reproducibility, limited sensitivity, and poorly understood reactions involved during analysis. Here, an LC-MS-based method for the sensitive and precise determination of total GalA contents in soluble and insoluble dietary fiber fractions of pectin containing samples is presented. The method is based on the degradation of GalA to the characteristic product 5-formyl-2-furancarboxylic acid (5FFA) in concentrated sulfuric acid under optimized conditions. The degradation product is extracted and quantified by UHPLC-ESI-MS. To compensate for degradation and extraction variability, the internal standard 13C6-galacturonic acid is used. Quantification is achieved by single ion monitoring (SIM) of 5FFA and the equivalent 13C-labeled degradation product. The validated method was successfully applied to various sample materials, including isolated galacturonic acid oligosaccharide standards with defined degrees of polymerization and plant-derived dietary fiber samples such as carrot, apple, and citrus pulp. Comparison to a widely used colorimetric assay demonstrated that the results of the two methods differ if applied to soluble fiber samples. Thus, the LC-MS approach represents a robust alternative to photometric assays, offering enhanced sensitivity, precision, and applicability for pectin analysis.
    Keywords:  5-Formyl-2-furancarboxylic acid; Chromatography-mass spectrometry; Colorimetric assays; Galacturonic acid; Pectins; Stable isotope dilution approach; Uronic acids
    DOI:  https://doi.org/10.1016/j.carbpol.2025.123816
  11. J Sep Sci. 2025 Jun;48(6): e70153
    Automatic Magnetic Dispersive Solid-Phase Extraction Combined With LC-MS/MS for the Rapid Quantitation of Six Gelsemium elegans Alkaloids in Biological Samples.
    Luyao Yang, Chunfang Ni, Chen Liang, Qianya Deng, Siyang He.
      In this study, an innovative, rapid, and automated method for quantifying six Gelsemium elegans alkaloids in biological samples using magnetic dispersive solid-phase extraction (MDSPE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Magnetic HLB extractant, NaH2PO4/Na2HPO4 buffer (0.2 M, pH 7) and acetonitrile were used in automatic MDSPE. The average pretreatment time was less than 2 min per sample. Atropine-d3 was employed as the internal standard (IS). The six alkaloids demonstrated a linear response (R2 > 0.997) in the concentration range of 1∼100 ng/mL and 2∼200 ng/g. Precision and accuracy were within ± 15%, and the extraction recovery rates for each alkaloid ranged from 60.32% to 105.32%. The samples were found to be stable under various conditions. In summary, we present the first automated MDSPE-LC-MS/MS method for simultaneous quantification of six Gelsemium elegans alkaloids (gelsemine, koumine, humantenmine, humantenine, humantenidine, and humantenirine) in multiple biological matrices (whole blood, urine, liver). Unlike prior studies focused on one to two analytes or single matrices, this method achieves unparalleled specificity for six alkaloids with detection limits up to 0.01 ng/mL (0.02 ng/g), addressing critical gaps in forensic toxicology for complex poisoning cases.
    Keywords:  Gelsemium elegans alkaloids; forensic toxicology; liquid chromatography tandem mass spectrometry; magnetic dispersive solid‐phase extraction
    DOI:  https://doi.org/10.1002/jssc.70153
  12. Methods Mol Biol. 2025 ;2925 161-172
    Polyamine Metabolism in Optic Nerve: Spatial Metabolomics with AP/MALDI Mass Spectrometry Imaging.
    Sean Meehan, Caleigh O'Connor, Eugene Moskovets, Konstantin Novoselov, Nivedita Bhattacharya, Sanjoy K Bhattacharya.
      Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is widely recognized as a leading method for studying spatial omics. Spatial multiomics platforms are most effective when they integrate multimodal datasets to provide a comprehensive understanding of biological contexts. By combining approaches like metabolomics and lipidomics, the throughput of spatial analysis methods can be significantly enhanced. In this particular protocol, polyamine pathway-related metabolites and lipids from a mouse optic nerve are imaged using atmospheric pressure MALDI.
    Keywords:  AP/MALDI; Lipids; Mass spectrometry imaging; OCT-embedded tissue; Ocular neurodegenerative disorders; Polyamines; amino acid
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_11
  13. Anal Bioanal Chem. 2025 Jun 07.
    LC-MS/MS-based simultaneous quantification of acylcarnitines, eicosapentaenoic acid, and docosahexaenoic acid: exploring potential biomarkers in human plasma.
    Baiba Gukalova, Kristaps Krims-Davis, Eduards Sevostjanovs, Aiga Leduskrasta, Ilze Konrade, Maija Dambrova, Edgars Liepinsh.
      Acylcarnitines have emerged as valuable markers of the intracellular fatty acid content, mitochondrial functionality, and fatty acid metabolism. However, acylcarnitines derived from polyunsaturated fatty acids (PUFAs) have not been previously examined. To address the need for monitoring lifestyle intervention and omega-3 supplementation studies, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), eicosapentaenoyl-L-carnitine (EPAC), and docosahexaenoyl-L-carnitine (DHAC). Matrix effects were normalized using a background subtraction approach, and the analytes were extracted from blood plasma via simple protein precipitation with acetonitrile. Chromatographic separation was achieved within 5 min on a reverse-phase C18 column using a gradient mobile phase composed of ammonium acetate and acetonitrile. The limits of quantification of the method were 2 μM for EPA/DHA and 2 nM for EPAC/DHAC. The method exhibited high precision and accuracy, with coefficient of variation and bias values less than 10%. The stability tests confirmed that the analytes remained stable under various conditions, such as remaining up to 6 h at room temperature and refrigeration and undergoing three freeze-thaw cycles; however, the acylcarnitines were unstable during long-term storage. This method is simple, fast, and cost-effective; thus, it is suitable for high-throughput analysis of samples from clinical studies. A PUFA supplement study in healthy volunteers revealed a more pronounced increase in plasma EPAC and DHAC levels than in EPA and DHA levels; these results indicated that PUFA-derived acylcarnitines were potential novel and sensitive markers of PUFA intake.
    Keywords:  Acylcarnitines; Docosahexaenoic acid; Docosahexaenoyl-L-carnitine; Eicosapentaenoic acid; Eicosapentaenoyl-L-carnitine; LC–MS/MS
    DOI:  https://doi.org/10.1007/s00216-025-05943-8
  14. Methods Mol Biol. 2025 ;2925 249-257
    Quantification of Melatonin with Immunoassay and Liquid Chromatography-Tandem Mass Spectrometry.
    Jonah Kutell, Avani Singal, Xing Du, Krishna Kishor.
      Melatonin, a key hormone in regulating circadian rhythms and sleep-wake cycles, presents challenges for quantification in C57BL/6 mice. C57BL/6 mice are the most used mouse strain for laboratory studies due to their inherent melatonin deficiency. This strain exhibits very low or undetectable melatonin levels in the pineal gland and blood, complicating standardization efforts in melatonin research. Here we present two primary techniques for melatonin detection in C57BL/6 mouse blood: radioimmunoassay (RIA), using the Novolytix (formerly Buhlmann) kit, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The Novolytix RIA kit offers high sensitivity with a detection limit below 0.3 pg/mL for a 1 mL sample, similar to mass spectrometric detection capability. LC-MS/MS, considered the gold standard, provides even greater sensitivity and specificity allowing for the detection of melatonin at levels as low as 1 pg/mL in mouse plasma. Both methods require careful sample preparation, including extraction steps to reduce matrix impurities and concentrate the melatonin in the sample. This protocol outlines detailed procedures for blood collection, sample preparation, and melatonin quantification using both RIA and LC-MS/MS methods; addressing the unique challenges posed by the low melatonin levels in C57BL/6 mice.
    Keywords:  HPLC; LC-MS/MS; Melatonin; Metabolite Detection; Pineal Gland; Plasma; Radioimmunoassay
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_18
  15. Front Pharmacol. 2025 ;16 1592731
    A sex-sensitive LC-MS/MS method with isotopic internal standard for prazosin bioequivalence: bridging precision medicine and generic drug policy in China.
    Hui Chen, Junping Guo, Zhenyu Zhu, Xiaomei Huang, Jincai Guo.
       Objective: To develop a rapid, sensitive, and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for prazosin quantification in human plasma, validate its application in bioequivalence studies, and investigate sex-specific pharmacokinetic differences in a Chinese population.
    Materials and methods: Plasma samples were processed by protein precipitation with methanol and analyzed using a Waters ACQUITY UPLC® HSS T3 column. Prazosin-d8 was used as an isotopic internal standard (IS) to enhance quantification accuracy. Chromatographic separation was performed with methanol (A) and 0.1% formic acid in water (B) as the mobile phases, using gradient elution at 0.35 mL/min. Quantification was achieved using positive ionization mode with multiple reaction monitoring (MRM) transitions of m/z 384.2→95.0 for prazosin and m/z 392.2→95.0 for IS.
    Results: The method demonstrated excellent linearity (0.1000-30.00 ng/mL, LLOQ: 0.1000 ng/mL), surpassing the sensitivity of prior methods. Bioequivalence analysis confirmed that the 90% confidence interval (CI) for AUC0-24, AUC0-∞, and Cmax geometric mean ratios fell within the regulatory acceptance range (90.00%-111.11%). Sex analysis revealed significantly higher AUC0-24 (+48%) and AUC0-∞ (+46%) medians in females (n = 4) than in males (n = 16) (P < 0.05), suggesting potential sex-based differences in prazosin pharmacokinetics.
    Conclusion: This study establishes the first LC-MS/MS method integrating isotopic IS and sex-specific pharmacokinetic profiling for prazosin, offering regulatory-compliant bioequivalence validation and insights into precision dosing strategies. These findings support China's generic drug policy and highlight the need for sex-stratified pharmacokinetic evaluations in bioequivalence assessments.
    Clinical trial registration number: ChiCTR2100050626.
    Keywords:  LC-MS/MS; bioequivalence; pharmacokinetics; prazosin; prazosin-d8
    DOI:  https://doi.org/10.3389/fphar.2025.1592731
  16. Methods Mol Biol. 2025 ;2925 309-328
    Reproducible Sectioning and Sample Preparation for Mass Spectrometry Imaging of Cancer Metabolism in Frozen Lung Tissue.
    Vanessa Y Rubio, Hayley D Ackerman, Nicole Hackel, Christina L Carr, Jaden Baldwin, John H Lockhart, Claudenir Ferrari, Jodi Balasi, Joseph O Johnson, Brooke Smedley, Eric Welsh, Oscar E Ospina, Paul A Stewart, Elsa R Flores, John M Koomen.
      Mass Spectrometry Imaging (MSI) for small molecules has emerged as the optimal technique to ascertain meaningful biology from spatial distributions of metabolites, lipids, and other classes of molecules. The success or failure of this approach rests on the sample preparation. Each tissue can have its unique challenges. In addition, the established histological processes for embedding frozen tissue, such as optimal cutting temperature medium, produce large polyethylene glycol clusters that suppress and interfere with the signal of biological molecules. To address these challenges, we provide a reproducible protocol for sectioning lung cancer tissue using a commercially available histological embedding matrix using tissues from genetically engineered mouse models of lung adenocarcinoma with a fluorescent reporter cassette to highlight additional microscopy methods used in parallel with mass spectrometry imaging to select regions of interest to compare tumor and adjacent lung tissue. These improvements to existing techniques produce high-quality sections of frozen tissue for histology and mass spectrometry imaging.
    Keywords:  Frozen Tissue; Lipids; Lung Cancer; Mass Spectrometry Imaging; Matrix-Assisted Laser Desorption Ionization (MALDI); Metabolomics; Tissue Sectioning
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_23
  17. Methods Mol Biol. 2025 ;2925 145-160
    Spatial Atmospheric Pressure MALDI HRMS Analysis of Histological Sections of FFPE Samples Reveals a Unique Subset of the Metabolome.
    Gilles Frache, David C Muddiman, Simona Ospitalieri, Caleigh R O'Connor, Dietmar R Thal, Peter D E M Verhaert.
      We describe a method that allows high-resolution mass spectrometry (HRMS) imaging of metabolites in tissue sections from formaldehyde-fixed, paraffin-embedded (FFPE) biobanks. This top-down variant of MS imaging expands the molecular scope of mass spectrometry histochemistry (MSHC) from peptidomics to metabolomics. The method makes the vast archives of FFPE biobanks accessible for MSHC-based biomarker discovery research of not only small endogenous peptides but also (a subset of) metabolites. FFPE biobank tissues include well-documented clinical samples representing diseases with a high medical need and often presently not clinically diagnosable and/or curable.Our protocol starts with FFPE tissue sections prepared from samples procured from biobanks. We describe how to remove paraffin and coat the section with MALDI matrix while maximally reducing analyte delocalization or washout. We detail appropriate programming of the MSHC data acquisition and illustrate a way to process MSHC data (including conversion to the generic imzML format) and browse MSHC datasets. Finally, we show options to present the data in the form of annotated MSHC images.
    Keywords:  Atmospheric pressure MALDI; Formaldehyde-fixed paraffin-embedded tissues; Mass spectrometry histochemistry; Orbitrap HRMS; Top-down mass spectrometry imaging
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_10
  18. Methods Mol Biol. 2025 ;2925 383-396
    NMR Metabolomics in Livestock: An Update of Current Methods and Applications.
    Claudia Torres-Calzada, Brian Lee, Mark Berjanskii, David S Wishart.
      As the global population increases, the livestock sector will face immense challenges to meet the demand for animal products. A more comprehensive, molecular-level understanding of livestock metabolism and physiology is essential to provide the key insights needed to enhance livestock productivity and health. In this regard, metabolomics, especially Nuclear Magnetic Resonance (NMR)-based metabolomics, offers a superb way to gain molecular-level understanding. NMR allows non-destructive analysis of biological samples without derivatization or chromatographic separation. Its high level of automation facilitates high-throughput analysis with minimal manual effort and very modest costs. Despite some limitations in sensitivity and sample volume requirements, NMR's exceptional reproducibility ensures consistent results across various experiments and laboratories. This chapter provides a comprehensive guide on preparing livestock samples from diverse biofluids and tissues for NMR analysis. It details the procedures for obtaining high-quality NMR spectra and describes spectral profiling techniques to identify and quantify water-soluble metabolites, leveraging metabolomics to enhance livestock productivity and health.
    Keywords:  Biofluids; High-throughput analysis; Livestock; Metabolomics; Nuclear Magnetic Resonance; Tissue; Water-soluble metabolites
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_25
  19. Methods Mol Biol. 2025 ;2925 25-29
    Analysis of Soybean Phospholipid Metabolites Using Triple Quadrupole Mass Spectrometry.
    Maria D Cabrera Gonzalez, Xing Du, Sanjoy K Bhattacharya.
      Phospholipids play an essential role in living cells, such as forming a lipid bilayer to create a barrier of protection against environmental changes and enabling cellular processes to occur in these compartments. Lipidomics is the study of fatty molecules within a biological system, their extracellular and cellular interactions, and the diseases to which the molecules contribute. Analytical chemistry is a common technique used to analyze these molecules, using a triple quadrupole mass spectrometer coupled to electrospray ionization (ESI) or heated electrospray ionization (H-ESI) mode. TSQ Quantum Access MAX Triple Quadrupole Mass Spectrometer provides many advantages when used for lipid analysis due to the numerous functions and adjustment of parameters allowing for a fast and accurate detection system of various molecules. This book chapter describes using the TSQ Quantum Access Max through direct infusion with a syringe pump to analyze phospholipids derived from soybeans.
    Keywords:  Lipid extraction; Lipidomics; Mass spectrometry; Phospholipids; Shotgun lipidomics; Triple quadrupole mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_2
  20. BBA Adv. 2025 ;7 100161
    DANGO: An MS data annotation tool for glycolipidomics.
    Masaaki Matsubara, Mayumi Ishihara, Michael Tiemeyer, Kazuhiro Aoki, René Ranzinger.
      Glycosphingolipids are essential components of all eukaryotic cells and play a major role in a broad range of cellular and biological processes, including growth, cell signaling, survival, differentiation, and disease. Glycosphingolipid structural diversity arises from heterogeneity in both the glycan and lipid moieties. Most currently available computational tools for annotating mass spectrometry data for glycosphingolipids primarily focus on glycan structure analysis, although a tool for annotating intact glycosphingolipids has recently been reported. Developing tools that integrate both glycan-centric analytical approaches and dynamic lipid composition changes, which influence functional membrane characteristics, would be highly beneficial. We have developed a glycosphingolipid computational tool, named DANGO (Data ANnotation system for GlycolipidOmics), for the automated annotation of glycolipidomic datasets. DANGO supports processing and annotation of mass spectrometry data to characterize both the glycan and lipid (ceramide) moieties (http://www.ms-dango.org/). DANGO annotates MS datasets using a glycosphingolipid database, which is created from a curated or user-defined glycan and ceramide collection, and proposes candidate structures to the user that match the experimental data. DANGO is implemented as an extension of GRITS Toolbox (http://www.grits-toolbox.org), employing functionalities such as display routines for post-processing and organized annotation of data and relevant metadata. Implementation of a novel filter algorithm in DANGO reduces false-positive identifications, resulting in enhanced reliability and shortened computational time for acquiring glycosphingolipid structural annotation. The labor-intensive manual annotation of mass spectrometry datasets has been the only approach to confident assignment of glycosphingolipid structural identity. DANGO provides intuitive workflows for enhancing the annotation of glycosphingolipidomic data.
    Keywords:  DANGO; Free software; Glycolipidomics; Glycosphingolipid; Mass spectrometry; Mass spectrometry data processing
    DOI:  https://doi.org/10.1016/j.bbadva.2025.100161
  21. Methods Mol Biol. 2025 ;2925 223-230
    LC-MS-Based Metabolic Fingerprinting of Vitreous Humor.
    Karolina Pietrowska, Diana Anna Dmuchowska, Joanna Godzien, Joanna Konopinska, Adam Kretowski, Michal Ciborowski.
      Vitreous humor (VH) is the largest structure in the eye that fills the space between the lens and the retina. Its composition and level of metabolites may reflect the physiological and pathophysiological processes occurring in the retina. A protocol for the preparation of VH samples for untargeted metabolomics was developed, and extracted metabolites were analyzed using liquid chromatography-quadrupole-time of flight-mass spectrometry (LC-QTOF-MS). Two types of chromatography were combined with mass spectrometry, reversed-phase (RP-LC-MS), and hydrophilic interaction (HILIC-LC-MS).
    Keywords:  HILIC chromatography; LC–MS; Reversed-phase chromatography; Untargeted metabolomics; Vitreous humor
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_15
  22. Bioinform Adv. 2025 ;5(1): vbaf100
    Enrichment analysis for spatial and single-cell metabolomics accounting for molecular ambiguity.
    Bishoy Wadie, Martijn R Molenaar, Lucas M Vieira, Theodore Alexandrov.
       Summary: Imaging mass spectrometry (imaging MS) has advanced spatial and single-cell metabolomics, but the reliance on MS1 data complicates the accurate identification of molecular structures, not being able to resolve isomeric and isobar molecules. This prevents application of conventional methods for overrepresentation analysis (ORA) and metabolite set enrichment analysis (MSEA). To address this, we introduce S2IsoMEr R package and a web app for METASPACE, which uses bootstrapping to propagate isomeric/isobaric ambiguities into the enrichment analysis. We demonstrate S2IsoMEr for single-cell metabolomics and the METASPACE web app for spatial metabolomics.
    Availability and implementation: METASPACE web app can be used on existing and new datasets submitted to METASPACE (https://metaspace2020.org). The source code for the S2IsoMEr R package is available on GitHub (https://github.com/alexandrovteam/S2IsoMEr).
    DOI:  https://doi.org/10.1093/bioadv/vbaf100
  23. Methods Mol Biol. 2025 ;2925 173-184
    Direct Colony Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Assay for Targeted Operon Metabolite Analysis.
    Nivedita Bhattacharya, Deepanwita Banerjee, Caleigh O'Connor, Dharmeshkumar Parmar, Eugene Moskovets, Peter D E M Verhaert, Anu Raghunathan, Venkateswarlu Panchagnula.
      Atmospheric pressure matrix-assisted laser desorption/ionization (AP/MALDI) mass spectrometry (MS) allows for highly sensitive and specific detection and relative quantification of metabolites of interest, with the key benefit of minimal sample preparation requirements for complex biological samples such as bacteria. Violacein is a violet-colored pigment produced by the bacterium Chromobacterium violaceum. In this chapter, we detail a high-throughput, high-resolution (HR) MS-based, targeted metabolite profiling protocol for analyzing violacein and its intermediates directly from bacterial colonies. This technique applies to a wide range of metabolites regulated by operon control and is suitable for a molecular phenotyping platform.
    Keywords:  AP/MALDI MS; Adaptive laboratory evolution (ALE); Antibiotic resistance; Chromobacterium violaceum; Metabolomics; Microbial analysis; Pathway metabolites; Vio operon
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_12
  24. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jun 03. pii: S1570-0232(25)00248-X. [Epub ahead of print]1263 124694
    Development and validation of a high-throughput LC-MS/MS bioanalytical method for the simultaneous quantification of cannabidiol and metabolites in human plasma.
    Ryan De Palma, Murali K Matta, Vikram Patel, Jeffry Florian, David G Strauss, Rodney Rouse.
      The removal of hemp from the definition of marijuana in the 2018 Agricultural Improvement Act has increased the number of cannabidiol-containing products available to consumers. Consequently, consumer use has also increased. Increased product availability and use drives the need for sensitive and specific analytical assays to measure the cannabidiol (CBD) and metabolites in patients, to establish dose-effect relationships and to gain knowledge of their pharmacokinetics. Here we describe the development and validation of a rapid high-throughput LC-MS/MS bioanalytical method for the quantification of cannabidiol and primary metabolites in human plasma to support an FDA-sponsored clinical study (NCT06192589). Sample preparation a single step protein precipitation followed by filtration through 96-well Phree™ Phospholipid removal plates. The method was validated over ranges of CBD: 1.95-500.00 ng mL-1; 7-hydroxy-cannabidiol (7-OH-CBD): 3.91-1000.00 ng mL-1; 7-carboxy-cannabidiol (7-COOH-CBD): 31.25-8000.00 ng mL-1. There was no cross-analyte interference, injection carryover, or matrix effect observed with this method. Analyte recoveries were consistent across three QC levels ranging from 83.90 to 90.85 %. Inter-day Accuracy across four QC level of all three analytes ranged from 93.87 to 107.31 % while precision ranged from 1.03 to 14.33 %. These results and other outlined in this manuscript met acceptance criteria as outline the current M10 Guidance for Bioanalytical Method Validation and Study Sample Analysis.
    Keywords:  Bioanalytical; Cannabidiol; High throughput; LC-MS/MS; M10 guidance; Metabolites; Method validation
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124694
  25. Methods Mol Biol. 2025 ;2925 297-308
    Untargeted Metabolomics of Human Embryonic Kidney (HEK) Cell Line.
    Xing Du, Claudia Gomez, Susanna Li, Emily Dorairaj, Samyak Jain, Sanjoy K Bhattacharya.
      Human embryonic kidney (HEK) cells' low maintenance and quick proliferation make it an ideal target to begin cellular research. Current literature on the metabolomic changes in the fatty acids synthesis (FAS) process is sparse. Thus, inhibition of the pathway in extensively researched HEK cells would provide further insight into upregulation and downregulation of metabolites. Liquid chromatography-mass spectrometry allows for highly sensitive quantification and qualification to provide a comprehensive understanding of the metabolomic changes that can be applied to specialized cell types.
    Keywords:  Fatty acids synthesis; HEK cells; Liquid chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_22
  26. Molecules. 2025 Jun 03. pii: 2451. [Epub ahead of print]30(11):
    An Innovative Strategy for Untargeted Mass Spectrometry Data Analysis: Rapid Chemical Profiling of the Medicinal Plant Terminalia chebula Using Ultra-High-Performance Liquid Chromatography Coupled with Q/TOF Mass Spectrometry-Key Ion Diagnostics-Neutral Loss Filtering.
    Jia Yu, Xinyan Zhao, Yuqi He, Yi Zhang, Ce Tang.
      Structural characterization of natural products in complex herbal extracts remains a major challenge in phytochemical analysis. In this study, we present a novel post-acquisition data-processing strategy-key ion diagnostics-neutral loss filtering (KID-NLF)-combined with ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) for systematic profiling of the medicinal plant Terminalia chebula. The strategy consists of four main steps. First, untargeted data are acquired in negative electrospray ionization (ESI-) mode. Second, a genus-specific diagnostic ion database is constructed by leveraging characteristic fragment ions (e.g., gallic acid, chebuloyl, and HHDP groups) and conserved substructures. Third, MS/MS data are high-resolution filtered using key ion diagnostics and neutral loss patterns (302 Da for HHDP; 320 Da for chebuloyl). Finally, structures are elucidated via detailed spectral analysis. The methanol extract of T. chebula was separated on a C18 column using a gradient of acetonitrile and 0.1% aqueous formic acid within 33 min. This separation enabled detection of 164 compounds, of which 47 were reported for the first time. Based on fragmentation pathways and diagnostic ions (e.g., m/z 169 for gallic acid, m/z 301 for ellagic acid, and neutral losses of 152, 302, and 320 Da), the compounds were classified into three major groups: gallic acid derivatives, ellagitannins (containing HHDP, chebuloyl, or neochebuloyl moieties), and triterpenoid glycosides. KID-NLF overcomes key limitations of conventional workflows-namely, isomer discrimination and detection of low-abundance compounds-by exploiting genus-specific structural signatures. This strategy demonstrates high efficiency in resolving complex polyphenolic and triterpenoid profiles and enables rapid annotation of both known and novel metabolites. This study highlights KID-NLF as a robust framework for phytochemical analysis in species with high chemical complexity. It also paves the way for applications in quality control, drug discovery, and mechanistic studies of medicinal plants.
    Keywords:  Terminalia chebula; UPLC-Q-TOF/MS; key ion diagnostics; neutral loss filtering; untargeted mass spectrometry
    DOI:  https://doi.org/10.3390/molecules30112451
  27. Anal Chim Acta. 2025 Aug 22. pii: S0003-2670(25)00609-9. [Epub ahead of print]1364 344215
    Development of a quantitative structure-response relationships to estimate concentrations of plasticizer metabolites in urine without reference standards using non-targeted analysis with liquid chromatography high-resolution mass spectrometry.
    Yong-Lai Feng, Jerrie Feng, Anca Baesu, Vanessa Martinez, Yan Li.
       BACKGROUND: Plasticizers are characterized as endocrine disruptors, and human exposure to these chemicals can adversely affect the reproductive system. Non-targeted analysis (NTA) using liquid chromatography - high resolution mass spectrometry (LC-HRMS) is a discovery-based approach for screening and identifying hundreds of compounds and metabolites in human samples without a priori knowledge of the chemical species that would be present in the sample. However, while NTA methods need to be standardized in order to provide reliable and reproducible qualitative results, there is also an urgent need to develop NTA methods that can provide quantitative information for identified compounds.
    RESULTS: We describe a quantitative structure-response relationship (QSRR) in NTA using LC-electrospray ionization (ESI)-HRMS that was established by combining retention behavior and structural descriptors. This relationship can be used to estimate the signal response factors of chemicals and we can utilize the estimated response factors to approximate the concentrations of plasticizer metabolites without reference standards. The model consists of 12 variables, being retention time and 11 molecular descriptors, that can estimate response factors of eleven testing chemicals with an error less than 30 %. The study demonstrated that the model can be updated with routine (e.g. daily) calibration curves and estimate concentrations of unknown metabolites in urine with an error less than 50 % for most compounds. The model can be applied to two different ionization systems without having a significant impact on its prediction accuracy.
    SIGNIFICANCE: While NTA provides the important information on identification of unknown and new chemicals/metabolites, it is crucial to provide quantitative concentrations for these compounds identified in NTA to support exposure assessment. Our approach enables a fast and simple estimation of the concentration of unknown metabolites of phthalates and their alternative chemicals without the need for respective reference standards. More importantly, the method is applicable to routine NTA studies and can be adapted to different LC-MS systems by simply updating the model with daily calibration data.
    DOI:  https://doi.org/10.1016/j.aca.2025.344215
  28. Anal Chim Acta. 2025 Sep 08. pii: S0003-2670(25)00650-6. [Epub ahead of print]1366 344256
    Advances in metallic nanostructures-assisted laser desorption/ionization mass spectrometry imaging of biological samples: A review.
    Joanna Nizioł, Sumi Krupa, Wiktoria Szuberla, Tomasz Ruman.
       BACKGROUND: Mass spectrometry imaging (MSI) has emerged as a powerful tool for the spatial visualization of biomolecules, driving advances in diverse fields such as biomedical research, plant metabolomics, and forensic science. Incorporating nanostructures, particularly metallic and metal oxide nanoparticles, has revolutionized laser desorption/ionization (LDI)-MSI by enhancing ionization efficiency, spatial resolution, and sensitivity.
    RESULTS: This review focuses on the preparation, application, and performance of various metallic nanostructures (e.g., gold, silver, platinum, and metal oxides) in LDI-MSI, emphasizing both fundamental physicochemical properties and their role in improving sensitivity, spatial resolution, and data reproducibility.
    SIGNIFICANCE: We provide a comparative assessment of metallic nanostructures versus other types of nanomaterials (quantum dots, carbon-based materials), highlight key advantages and current limitations, and offer a roadmap for future developments in nanomaterial-assisted MSI, including prospective strategies for stabilizing and functionalizing surfaces, exploring alternative laser wavelengths, and ensuring robust analytical workflows.
    Keywords:  LDI-MS; MSI; Mass spectrometry; Mass spectrometry imaging; Metallic nanostructures; Nanoparticles
    DOI:  https://doi.org/10.1016/j.aca.2025.344256
  29. bioRxiv. 2025 Jun 01. pii: 2025.05.28.656653. [Epub ahead of print]
    Neural Spectral Prediction for Structure Elucidation with Tandem Mass Spectrometry.
    Runzhong Wang, Mrunali Manjrekar, Babak Mahjour, Julian Avila-Pacheco, Joules Provenzano, Erin Reynolds, Magdalena Lederbauer, Eivgeni Mashin, Samuel Goldman, Mingxun Wang, Jing-Ke Weng, Desirée L Plata, Clary B Clish, Connor W Coley.
      Structural elucidation using untargeted tandem mass spectrometry (MS/MS) has played a critical role in advancing scientific discovery [1, 2]. However, differentiating molecular fragmentation patterns between isobaric structures remains a prominent challenge in metabolomics [3-10], drug discovery [11-13], and reaction screening [14-17], presenting a significant barrier to the cost-effective and rapid identification of unknown molecular structures. Here, we present a geometric deep learning model, ICEBERG, that simulates collision-induced dissociation in mass spectrometry to generate chemically plausible fragments and their relative intensities with awareness of collision energies and polarities. We utilize ICEBERG predictions to facilitate structure elucidation by ranking a set of candidate structures based on the similarity between their predicted in silico MS/MS spectra and an experimental MS/MS spectrum of interest. This integrated elucidation pipeline enables state-of-the-art performance in compound annotation, with 40% top-1 accuracy on the NIST'20 [M+H] + adduct subset and with 92% of correct structures appearing in the top ten predictions in the same dataset. We demonstrate several real-world case studies, including identifying clinical biomarkers of depression and tuberculous meningitis, annotating an aqueous abiotic degradation product of the pesticide thiophanate methyl, disambiguating isobaric products in pooled reaction screening, and annotating biosynthetic pathways in Withania somnifera . Overall, this deep learning-based, chemically-interpretable paradigm for structural elucidation enables rapid molecular annotation from complex mixtures, driving discoveries across diverse scientific domains.
    DOI:  https://doi.org/10.1101/2025.05.28.656653
  30. Cell. 2025 Jun 09. pii: S0092-8674(25)00565-3. [Epub ahead of print]
    The microbiome diversifies long- to short-chain fatty acid-derived N-acyl lipids.
    Helena Mannochio-Russo, Vincent Charron-Lamoureux, Martijn van Faassen, Santosh Lamichhane, Wilhan D Gonçalves Nunes, Victoria Deleray, Adriana V Ayala, Yuichiro Tanaka, Abubaker Patan, Kyle Vittali, Prajit Rajkumar, Yasin El Abiead, Haoqi Nina Zhao, Paulo Wender Portal Gomes, Ipsita Mohanty, Carlynda Lee, Aidan Sund, Meera Sharma, Yuanhao Liu, David Pattynama, Gregory T Walker, Grant J Norton, Lora Khatib, Mohammadsobhan S Andalibi, Crystal X Wang, Ronald J Ellis, David J Moore, Jennifer E Iudicello, Donald Franklin, Scott Letendre, Loryn Chin, Corinn Walker, Simone Renwick, Jasmine Zemlin, Michael J Meehan, Xinyang Song, Dennis Kasper, Zachary Burcham, Jane J Kim, Sejal Kadakia, Manuela Raffatellu, Lars Bode, Hiutung Chu, Karsten Zengler, Mingxun Wang, Dionicio Siegel, Rob Knight, Pieter C Dorrestein.
      N-Acyl lipids are important mediators of several biological processes including immune function and stress response. To enhance the detection of N-acyl lipids with untargeted mass spectrometry-based metabolomics, we created a reference spectral library retrieving N-acyl lipid patterns from 2,700 public datasets, identifying 851 N-acyl lipids that were detected 356,542 times. 777 are not documented in lipid structural databases, with 18% of these derived from short-chain fatty acids and found in the digestive tract and other organs. Their levels varied with diet and microbial colonization and in people living with diabetes. We used the library to link microbial N-acyl lipids, including histamine and polyamine conjugates, to HIV status and cognitive impairment. This resource will enhance the annotation of these compounds in future studies to further the understanding of their roles in health and disease and to highlight the value of large-scale untargeted metabolomics data for metabolite discovery.
    Keywords:  HIV; MASST; MassQL; N-acyl lipids; SCFA; metabolomics data mining; microbial; neurocognitive impairment; repository-scale analysis; short-chain fatty acids
    DOI:  https://doi.org/10.1016/j.cell.2025.05.015
  31. Anal Bioanal Chem. 2025 Jun 13.
    Multi-residue environmental method for seed treatment insecticides, neonicotinoid degradation products, and fungicides using liquid chromatography tandem mass spectrometry with a new ionization source.
    Jascika A A Maclean, Shannon Bartelt-Hunt, Joyce Cristale, Sathaporn Onanong, Daniel D Snow.
      Few methods provide simultaneous determination of multiple pesticides and degradation products in environmental samples using liquid chromatography tandem mass spectrometry (LC-MS/MS). As LC-MS/MS method performance is significantly influenced by the type and design of the ion source, we compared three ion sources: electrospray ionization (ESI) source, atmospheric pressure chemical ionization (APCI) source and UniSpray™ ionization source. A gain in sensitivity was observed with UniSpray™ and ESI as compared to APCI source on the same instrument. Matrix effects in the three interfaces were evaluated in reagent water and wastewater extracts. UniSpray™ showed the lowest matrix effect among the three sources, with APCI exhibiting more pronounced signal enhancement. A solid-phase extraction method using the UniSpray™ source provides method detection limits (MDLs) ranging from 0.00189 to 0.0209 µg/L in extracts from water samples. Recoveries in water ranged up to 94.35%, with above 60% of the pesticides having an average recovery exceeding 70%.
    Keywords:  Electrospray ionization; Mass spectrometry; Neonicotinoids; UniSpray
    DOI:  https://doi.org/10.1007/s00216-025-05927-8
  32. Nat Protoc. 2025 Jun 12.
    High-spatial-resolution mass spectrometry imaging of biological tissues using a microfluidic probe.
    Li-Xue Jiang, Xiangtang Li, Matthias Polack, Detlev Belder, Julia Laskin.
      Nanospray desorption electrospray ionization (nano-DESI) is a liquid extraction-based ambient ionization mass spectrometry imaging (MSI) technique that enables quantitative molecular mapping of biological samples in their native state with high spatial resolution. To facilitate the wider adoption of nano-DESI MSI by the scientific community, we have developed a robust and user-friendly microfluidic probe (MFP). The probe has been used to achieve high spatial resolution of 8-10 µm and up to 10-fold improvement in the experimental throughput, enabling imaging of large tissue sections with cellular resolution. Here, we provide detailed instructions for designing, fabricating and operating MFPs. In addition, we describe a complete workflow for nano-DESI MSI, covering every step from probe assembly to data acquisition and analysis. Although the fabrication of MFPs requires expertise in microfluidics and can take a few days, the process can be outsourced to qualified companies for manufacturing. Once the MFP is fabricated, the entire imaging workflow can be completed in several hours, depending on the sample size. For example, a sample with an area of 1 cm² can be analyzed in <10 h at a spatial resolution of 10 µm. The exceptional performance and ease of use of these probes will make high-resolution nano-DESI MSI more accessible to the scientific community.
    DOI:  https://doi.org/10.1038/s41596-025-01188-y
  33. Biomed Chromatogr. 2025 Jul;39(7): e70145
    A New, Fast, and Sensitive UPLC-ESI-MS/MS Method for the Simultaneous Analysis of 13 Neurotransmitters in Rat Brain.
    Ahmad A Deeb, Alaa M Hammad, Sawsan I Khdair, Hamza Abumansour.
      Neurotransmitters are crucial for the chemical signaling in the central nervous system. The present work proposes a novel UPLC-ESI-MS/MS method for simultaneously determining 13 neurotransmitters in the amygdala brain region in rats. A Zorbax SB C18 column and a gradient mobile phase consisting of a mixture of water and acetonitrile comprising 0.2% heptafluorobutyric acid and 0.1% formic acid were used. By the ICH guideline, outstanding linearity of the method (R2 > 0.99), detectors' sensitivity, and specificity are demonstrated with low LOD (0.011 pg/mL for endorphins to 2.69 ng/mL for serotonin) and LOQ (0.06 for endorphins to 14 ng/mL for serotonin) values. The recovery ranged from 94% for anandamide to 98% for fatty acid amide hydrolase. Following the static test validation process, aspects of the method such as accuracy, precision, matrix effect, and stability were confirmed, with each neurotransmitter assigned retention times ranging from 1 to 4.3 min (GABA and endorphins, respectively). In the context of brain regions, the technique allows comprehensive neurochemical analysis in the amygdala and is a step forward for neurochemistry understanding. It opens up a brand-new approach to studying brain disorders, providing brain operation and its pathologies in an elaborate matrix in an extremely short time.
    Keywords:  UPLC‐ESI‐MS/MS; amygdala; neurological disorders; neurotransmitters; rat brain
    DOI:  https://doi.org/10.1002/bmc.70145
  34. Anal Chim Acta. 2025 Aug 22. pii: S0003-2670(25)00622-1. [Epub ahead of print]1364 344228
    Utilizing the total phosphorus content of lipid extracts as a normalization strategy in quantitative lipidomics studies.
    Johannes Scholz, Anna Gremme, Julia Hillebrand, Jasper Baldauf, Lennart J Bendheuer, Jonas Berreiter, Anne C Sehnal, Julia Bornhorst, Heiko Hayen.
       BACKGROUND: Normalization is a crucial element of quantitative lipidomics studies, for which there is still no uniform recommended strategy. There are various approaches for normalizing samples, including referencing to the total volume or weight, to the DNA content or the protein content. In addition, samples can be normalized to the phosphorus content, but either not all phospholipid classes are included or a less sensitive colorimetric assay is used. It is therefore necessary to develop new ways of normalization that are both specific and sensitive to compensate for the biological sample variance and the variance from pre-analytical and analytical steps.
    RESULTS: We present a tailored normalization method for lipid quantification which is based on the total phosphorus content (TPC) of lipid extracts. The TPC reflects the amount of phospholipids present in the sample, because polar phosphorus-containing compounds like DNA and phosphorylated sugars are separated due to the extraction procedure. In order to test the normalization strategy, the (phospho)-lipids in the model organism C. elegans were quantified using SFC-TIMS-MS/MS. Subsequently, a digestion method was developed to degrade the lipids present in the organic extract. This was used to determine the TPC via ICP-OES and ICP-MS/MS analysis. In particular, very low detection limits (LOQ = 0.3 μg L-1) can be achieved with the ICP-MS/MS method. The elemental and molecular information were then combined, and the samples were normalized to TPC. The normalization showed a significant improvement in the standard deviations compared to the initial concentrations as well as an improvement compared to the normalization based on the total protein content, which is very frequently used.
    SIGNIFICANCE: Our method shows a sensitive way to determine the TPC in total lipid extracts of various samples, exemplified for human plasma and nematode samples. The normalization minimizes the standard deviations in biological samples even in comparison with other normalization approaches, i.e. protein content, and can contribute to increasing the conclusiveness of quantitative lipidomics studies.
    Keywords:  Caenorhabditis elegans; ICP-MS; ICP-OES; Lipidomics; Normalization; SFC-TIMS-MS
    DOI:  https://doi.org/10.1016/j.aca.2025.344228
  35. Chirality. 2025 Jun;37(6): e70037
    Quantitative Chiral Separations on KRAS Inhibitor Sotorasib by UHPLC-MS/MS: Method Validation and Enantioselective Pharmacokinetics Assessment in Rat Plasma.
    Jony Susanna Kandula, Keyur Parmar, A R Priyadharshni, Abhay Kumar, P Radhakrishnanand.
      In this research, estimation of enantioselective pharmacokinetics and biological interconversion of sotorasib were investigated. This is the first report for the novel chiral liquid chromatography-tandem mass spectrometry method for the enantioselective pharmacokinetics determination in biological matrix. Attempts were made to achieve separation in reverse phase mode with mass compatible mobile phase. The baseline chiral separation was achieved with a mobile phase consisting of acetonitrile and aqueous ammonium bicarbonate (15 mM; 95/5 v/v) on Chiralpak IC (250 × 4.6 mm, 5 μm). Positive electrospray ionization was used for monitoring multiple reactions by triple quadrupole. The developed bioanalytical method was validated for each SOT enantiomer, assessing selectivity and specificity, linearity, accuracy, precision, recovery, matrix effect, dilution integrity, and stability. The lower limit of quantification was identified as 1 ng/mL for both SOT enantiomers. The results were found to be in compliance with the bioanalytical method validation guideline. The preclinical study revealed the disparate pharmacokinetic profile between the (R) and (S)-sotorasib, after single oral administration (10 mg/kg) to rats. This is the first investigational evidence of enantioselective behavior of sotorasib in vivo. The results demonstrated that lower maximum concentration, area under curve, and higher clearance and volume of distribution for (R)-sotorasib reveal the poorer bioavailability and higher elimination rate. The study provides insights in controlling the enantiomeric impurity in quality control aspects. This research also provides a reference for clinical practice and supports further research in distomer toxicity, enantioselective drug metabolism, and drug interactions.
    Keywords:  biological interconversion; chiral bioanalysis; enantioselective pharmacokinetics; liquid chromatography–tandem mass spectrometry; method validation
    DOI:  https://doi.org/10.1002/chir.70037
  36. Anal Chim Acta. 2025 Aug 22. pii: S0003-2670(25)00618-X. [Epub ahead of print]1364 344224
    A fully automated, high-throughput electro-extraction and analysis workflow for acylcarnitines in human plasma and mouse muscle tissues.
    Yupeng He, Paul Miggiels, Amy Harms, Yvonne Rijksen, Renata M C Brandt, Wilbert P Vermeij, Bert Wouters, Thomas Hankemeier.
       BACKGROUND: The labor-intensive and time-consuming nature of sample preparation poses significant challenges for bioanalysis, especially for large-scale samples characterized by limited volumes/mass, and low analyte abundance. Additionally, manual sample processing can compromise reproducibility. To overcome these limitations, automation and high-throughput methodologies are essential, highlighting the need for an automated, high-throughput sample preparation and analysis workflow.
    RESULTS: This study presents a fully automated, high-throughput electro-extraction (EE) platform integrated with a CTC PAL3 autosampler and liquid chromatography-mass spectrometry analyzer. The integrated platform underwent qualification, followed by optimization of EE parameters using a Design of Experiment approach. Ten acylcarnitines were selected as model analytes. The optimization models exhibited strong fits (p < 0.006, R2 > 0.91). The optimized platform achieved an enrichment factor of up to 400 (an extraction recovery of up to 99 %) in designed academic samples, and was effectively implemented and evaluated using 20 μL of spiked human plasma samples. To test clinically relevant materials, the platform was utilized to study the effects of muscle tissue isolation speed on acylcarnitine stability, and to examine acylcarnitine abundance across muscle types in progeria (sarcopenia) mouse muscle. We found that the speed of muscle isolation does not affect measured levels of acylcarnitines, and detected higher acylcarnitine abundances are consistent with literature.
    SIGNIFICANCE: This study provides an automated, high-throughput, and cost-effective workflow enabling extraction and analysis of 120 samples per day, with a cost of <0.1 Euro per sample. It presents a significant stride towards the creation of fully-automated, high-throughput bioanalysis workflows for large-scale studies involving biomass limited samples in the foreseeable future.
    Keywords:  Automation; Bioanalysis; Electro-extraction; High-throughput; Sample-preparation; Sarcopenia
    DOI:  https://doi.org/10.1016/j.aca.2025.344224
  37. Anal Chim Acta. 2025 Aug 22. pii: S0003-2670(25)00621-X. [Epub ahead of print]1364 344227
    Cytokine profiling in COVID-19 patients by using ion mobility-mass spectrometry-based parallel reaction monitoring.
    Ling Yan, Chenrui Yuan, Runhou Zhou, Li Zhong, Kelvin Kai-Wang To, Na Liu, Xin Diao, Guangshan Xie, Hongzhi Zhao, Haijiang Wu, Lin Zhu, Zhiwei Chen, Zongwei Cai.
      Cytokine therapy, a non-antigen-specific strategy, has led to several FDA-approved drugs. Given the role of dysregulated cytokine expression in diseases such as COVID-19, accurate quantification is critical in both clinical and research settings. While antibody-based assays offer high sensitivity, their reliance on specific antibodies limits multiplexing and increases analytical complexity. Conversely, mass spectrometry methods like multiplexed reaction monitoring provide higher throughput but lack the sensitivity to detect physiological cytokine levels and the resolution to distinguish structural isomers. Thus, a new MS-based approach is needed that integrates high sensitivity with the ability to resolve structurally similar cytokines. We developed an ion mobility-mass spectrometry (IM-MS)-based parallel reaction monitoring (PRM) method to establish the first Cytokine Ion Mobility Peptide (CIMP) databank and enable high-throughput cytokine profiling in serum samples from COVID-19 patients. By introducing ion mobility as an additional gas-phase separation dimension alongside liquid chromatography, the method enhances analyte resolution based on structural differences, facilitating the separation of isomers within the ion mobility trap. The incorporation of ion mobility as a complementary separation parameter enables the distinction of homologous cytokines and structural isomers (e.g., IFNA1/IFNA2, IFNL1/IFNL3, and peptide isomers), which remains challenging for conventional antibody-based assays. The method achieved a limit of detection of 62.9 fmol/L and a limit of quantification of 210 fmol/L across 31 cytokines, demonstrating greater sensitivity than traditional multiple reaction monitoring (MRM) approaches and enabling quantification at physiological concentration levels, assuming comparable background signal across platforms. The IM-MS-PRM method offers a multiplexed, high-throughput, and adaptable platform that eliminates the need for multiple assays while delivering excellent reproducibility. It enables accurate and sensitive cytokine quantification from minimal volumes of COVID-19 patient serum. Combined with the CIMP databank, this approach allows precise differentiation between early and late severe COVID-19 cases, supporting improved diagnostic and therapeutic decision-making.
    Keywords:  COVID-19; Cytokine profiling; Ion mobility; Label-free; Parallel reaction monitoring
    DOI:  https://doi.org/10.1016/j.aca.2025.344227
  38. Methods Mol Biol. 2025 ;2925 185-201
    Metabolomics of the Kynurenine Pathway by Laser Desorption Ionization Mass Spectrometry (LDI-MS).
    Pallavi Lahiri, S Krishnaraj, Priyakshi Gogoi, Ambalika Roy, Dipankar Ghosh.
      The kynurenine (KYN) pathway is a branch of tryptophan metabolic cascade. The KYN pathway is unique since it generates multiple neuroactive metabolites, including 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA), quinolinic acid (QUIN), and picolinic acid (PIC). Whereas KYNA and PIC are neuroprotective, 3-HK and QUIN are potently neurotoxic and attribute to major neurodegenerative diseases like schizophrenia, Alzheimer's disease, Huntington's disease, bipolar disorder, and depression. It is increasingly evident that the ratio(s) between the neurotoxic versus neuroprotective metabolites may help predict the manifestations of disease versus health. Therefore, high-throughput platforms for determining the relative levels of these kynurenine metabolites in biofluids offer considerable potential. Current analytical tools for studying the KYN pathway include assays of branching enzymes, PCR, immunoanalysis, and liquid chromatography-mass spectrometry. Neither of these offers high-throughput, cost-effective analyses suited for clinical or drug-screening applications. In this report, a Laser Desorption Ionization Mass Spectrometry (LDI-MS) method is described using SBA-15 mesoporous silica. The system allows fast, high-resolution relative quantitation of neurotoxic kynurenines using targeted metabolomics on conventional Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (mass spectrometry) (MALDI-TOF) platforms.
    Keywords:  Kynurenic acid (KYNA); Kynurenine pathway; LDI-MS; Neuroactive metabolites; Relative Quantitation; Tryptophan metabolism
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_13
  39. Anal Chem. 2025 Jun 13.
    Controllable Ion-Repulsion Enables Rapid and Trace-Level Detection of Hydrophobic Sulfonylurea Herbicides with ICPMS/MS without the Organic Mode.
    Bassam Lajin, Walter Goessler.
      The inductively coupled plasma tandem mass spectrometry (ICPMS/MS) is emerging as a detector for compounds tagged with a nonmetal, but can be hampered by poor compatibility with organic eluents. Herein, we describe an alternative approach to using high fractions of organic solvents for the detection of hydrophobic compounds while presenting a novel sulfur speciation application involving the determination of a common and widely used class of hydrophobic sulfonylurea herbicides. The approach is based on controllable retention using an ion-repulsion concept, and it is shown that this approach enables minimizing the amount of organic eluent needed for speciation analysis, eliminating the need for the organic ICPMS mode and therefore achieving uncompromised sensitivity. The applied approach enabled fast analysis within 8 min with a mobile phase containing as little as 10% methanol. The limit of detection was 0.3 μg S L-1 (injection volume 20 μL), which is on par with the lowest achievable LOD for sulfur speciation with ICPMS/MS detection, highlighting the utility of the ion-repulsion approach in overcoming the current limitations of the organic ICPMS mode commonly used with ICPMS detection. Spiking experiments showed the developed method to be directly applicable to a river water matrix with no need for sample preparation or internal standards unlike currently used methods based on ESIMS/MS detection. The ion-repulsion concept is generally applicable to speciation analysis with ICPMS detection for ionizable analytes, provides similar control over retention to organic solvents, and aids in avoiding the limitations of the organic ICPMS mode.
    DOI:  https://doi.org/10.1021/acs.analchem.5c01663
  40. J Chromatogr A. 2025 Jun 05. pii: S0021-9673(25)00473-X. [Epub ahead of print]1757 466127
    Simultaneous determination of 12 fluorescent whitening agents in paper and plastic food packaging materials for infants by high-performance liquid chromatography-tandem mass spectrometry.
    Deng Hong, Qili Hu, Jianbo Hou, Yi Ruan, Ting Liu, Wen Xie, Liangli Wang, Yan Li.
      A rapid screening method was developed to simultaneous determine 12 fluorescent whitening agents (FWAs) in paper and plastic food packaging materials for infants by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was ultrasonically extracted by the mixed solvent of tetrahydrofuran-40 % acetonitrile aqueous solution. Separation was performed on Eclipse XDB-C18 chromatographic column (150 mm×4.6 mm, 5 μm) by gradient elution with acetonitrile/5 mmol/L ammonium acetate aqueous solution as the mobile phase. Quantitative detection was performed in the multiple reaction monitoring (MRM) mode under the electric spray ionization (ESI) source and positive/negative ion automatic switching scanning mode. Retention time and ion abundance ratio were retained for qualitative analysis and external standard method were used for quantitative analysis. The results indicated that the 12 FWAs showed good linearities within a certain concentration range, with a correlation coefficient greater than 0.998. The detection limits of the method were 0.008-0.2 mg/kg, and the lower limits of quantification were 0.02-0.6 mg/kg. In the paper and plastic food packaging materials, the concentration spiking of three levels were conducted. The recoveries for paper were 89.0 %-111 %, with relative standard deviations (RSD) of 1.7 %-8.1 %. The recoveries for plastic were 82.8 %-108 % with RSD of 1.4 %-9.7 %. This simple pre-treatment, accurate results, high sensitivity and high selectivity method can be used for qualitative screening and quantitative analysis of FWAs in paper and plastic packaging materials for infants.
    Keywords:  Fluorescent whitening agents; Food packaging materials for infants; HPLC-MS/MS; Paper; Plastic
    DOI:  https://doi.org/10.1016/j.chroma.2025.466127
  41. bioRxiv. 2025 May 26. pii: 2025.05.25.655991. [Epub ahead of print]
    Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD + metabolites in CD38 knockout mice exhibiting improved metabolism.
    Charles A Schurman, Joanna Bons, Prasanna Vadhana Ashok Kumaar, Jingji Fang, Andrea Roberts, Genesis Vega Hormazabal, Rebeccah R Riley, Nannan Tao, Eric Verdin, Birgit Schilling.
      Mass spectrometry imaging (MSI) is a rapidly advancing technology that provides mapping of the spatial molecular landscape of tissues for a variety of analytes. Matrix-assisted laser desorption/ionization (MALDI)-MSI is commonly employed, however, confident in situ identification and accurate quantification of analytes remain challenging. We present a novel imaging methodology combining trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) with MALDI ionization for targeted imaging parallel reaction monitoring (iprm-PASEF). We investigated the spatial distribution of lipids and metabolites in liver tissues from wild-type and CD38 knockout mice (CD38 -/- ). CD38, an enzyme involved in nicotinamide adenine dinucleotide (NAD + ) metabolism, significantly influences liver metabolic function and contributes to age-related NAD + decline. Although CD38 deletion previously was linked to improved metabolic phenotypes, the underlying spatial metabolic mechanisms are poorly understood. The spatial iprm-PASEF workflow enabled confident identification and differentiation of lipid isomers at the MS2 fragment ion level and revealed increased NAD + and decreased adenosine diphosphate ribose (ADPR), a by-product of NAD + hydrolysis, in CD38 -/- livers. This approach provided confident, specific, and robust MS2-based identification and quantification of fragment ions in spatial MSI experiments. Additionally, the innovative iprm-PASEF opens unprecedented opportunities for spatial metabolomics and lipidomics, offering spatially resolved insights into molecular mechanisms.
    DOI:  https://doi.org/10.1101/2025.05.25.655991
  42. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2025 Jun 09. 41 e20250009
    Bioanalytical Method Development and Validation of Dapagliflozin in Human Plasma Using RP-HPLC Method.
    Pravin Rangnath Dighe, Manoj Ramesh Kumbhare.
       BACKGROUND: Dapagliflozin is used for controlling blood glucose levels in patients with type 2 diabetes. It is a sodium-glucose cotransporter 2 inhibitor, which enhances the elimination of blood glucose through the urine by inhibiting the protein involved in the transport mechanism of SGLT2. Dapagliflozin requires a selective and sensitive bioanalytical RP-HPLC method.
    AIM: Reverse phase - high performance liquid chromatography technique was used to develop and validate a bioanalytical method for the quantification of dapagliflozin (DAPA) in human plasma.
    METHODS: The internal standard (IS) used was azilsartan medoxomil. In isocratic mode, the mobile phase consisted of 50:50 v/v acetonitrile and 0.1% orthophosphoric acid in water at a flow rate of 1.0 mL/min. The chromatogram was recorded at 224 nm. For the chromatographic separation, a Kromasil C18 column (250 mm × 4.6 mm; 5μ) was used. The drug was extracted from plasma samples by the protein precipitation method.
    RESULT AND DISCUSSION: The chromatographic run time was 15 min. Dapagliflozin and IS eluted at 4.6 and 5.7 min, respectively. The method was selective and sensitive, with a limit of quantification of 1.50 µg/mL. The developed method was found to be linear in the range of 1.50-60 µg/mL (r2 = 0.9994). The accuracy and precision obtained from six sets of quality control (QC) samples ranged from 96.23% to 108.67% and 1.35% to 3.19%, respectively. The extraction recovery of dapagliflozin in three QC samples ranged from 87.39% to 90.78%. The bench-top stability, stock solution stability, stability of processed extracted samples at room temperature, and freeze-thaw stability evaluations showed no evidence of degradation of dapagliflozin.
    CONCLUSION: The stability, selectivity, sensitivity, and reproducibility of the developed method make it suitable for the determination of dapagliflozin in human plasma.
    Keywords:  Dapagliflozin; Internal standard; Protein precipitation; RP-HPLC; Recovery
    DOI:  https://doi.org/10.62958/j.cjap.2025.009
  43. J Sep Sci. 2025 Jun;48(6): e70191
    High-Temperature Liquid Chromatography of Isomeric Organochlorine Herbicides and Their Degradation Products.
    Michal Kašpar, Petra Peroutková, Jiří Palarčík, Petr Česla.
      Alachlor, acetochlor, and metolachlor are organochlorine herbicides extensively used for weed control in agricultural practices, yet their use is prohibited within the European Union due to environmental and health concerns. These compounds and their degradation products exhibit high environmental persistence, necessitating reliable and rapid analytical methods for monitoring. Conventional reversed-phase liquid chromatography employing C18 columns is commonly utilized for their analysis. However, alkyl silica columns, and especially C18 columns, are not suitable for the effective separation of isomers, necessitating the integration of tandem mass spectrometry for enhanced analytical accuracy. This study explores the application of a porous graphitic carbon-based column to overcome these limitations. High-temperature chromatography enabled improved efficiency and selectivity in separating the target compounds. The influence of temperature, flow rate, pH, and mobile phase composition on the retention of alachlor, acetochlor, metolachlor, and their degradation products was systematically evaluated. A method suitable for separation and quantification of the herbicides was developed, highlighting the potential of porous graphitic carbon columns for robust and precise analysis in complex environmental matrices.
    Keywords:  chloracetanilide herbicides; diastereomers; isomers; pesticides; porous graphitic carbon
    DOI:  https://doi.org/10.1002/jssc.70191
  44. Anal Bioanal Chem. 2025 Jun 10.
    An LC/MS/MS method for quantifying 25-hydroxyvitamin D3 in finger-prick plasma sample prepared by DEMECAL® micro plasma separation device: toward mail-in assessment of vitamin D status.
    Kodai Maeda, Kyoka Sakamoto, Eiko Ito, Shinya Sugimoto, Shingo Tajima, Takahisa Sasahara, Shunji Kawamura, Kazuo Yaegashi, Tatsuya Higashi.
      A mail-in assessment of vitamin D status will make it possible for the subjects to early detect any vitamin D insufficiency/deficiency at their homes, thus leading to a better management of their health. In this study, a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for quantifying 25-hydroxyvitamin D3 [25(OH)D3], which is the established marker of the vitamin D status, in the finger-prick blood plasma sample prepared by the DEMECAL® device. By using the DEMECAL® device, the subjects can collect their finger-prick blood at their homes and immediately separate the plasma from the blood by themselves. The DEMECAL®-based procedure had advantages over the venipuncture- and dried blood spot-based procedures because it is less invasive and a medical professional-free blood collection, and no need to be concerned with the heterogeneity of the analyte distribution in the sample. The developed LC/ESI-MS/MS method enabled the precise and accurate quantification of 25(OH)D3 and provided the lower limit of quantification of 0.10 ng/mL in the diluted plasma sample due to the derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione. 25(OH)D3 was stable in the plasma sample prepared by the DEMECAL® device at room temperature (23-27 °C) for 7 days (simulated condition in the mail) and at 4 °C for 30 days (simulated condition during storage at a laboratory). The measured 25(OH)D3 concentrations by the DEMECAL®-based procedure well agreed with those by the conventional venipuncture-based procedure. These results demonstrated that the derivatization-LC/ESI-MS/MS combined with the DEMECAL®-based sample preparation is a promising procedure for the mail-in assessment of the vitamin D status.
    Keywords:  Derivatization; Finger-prick blood; LC/ESI–MS/MS; Micro plasma separation; Vitamin D status
    DOI:  https://doi.org/10.1007/s00216-025-05939-4
  45. Environ Toxicol Chem. 2025 Jun 11. pii: vgaf151. [Epub ahead of print]
    Targeted Quantitation of 6ppd-Quinone in Fish Tissue Samples with Liquid Chromatography-Tandem Mass Spectrometry.
    Adam H Moody, David J Soucek, David A Alvarez.
      The tire additive transformation product N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) has recently garnered global attention due to its acute toxicity to some salmonids, such as coho salmon (Oncorhynchus kisutch), and its ubiquitous presence in urban stormwater systems. In the present study, we developed and compared the extraction efficiency of two sample preparation methodologies for quantification of 6PPD-quinone among two fish tissue sample types that included fillet of smallmouth bass (Micropterus dolomieu) and whole-body samples of O. kisutch fry subjected to in vivo exposure tests with 6PPD-quinone. The two sample preparation methods tested included an accelerated solvent extraction (ASE) approach and a sonication extraction approach. Both sample preparation methods included identical purification steps for the crude sample extracts with enhanced matrix removal cartridges. The purified sample extracts were subjected to targeted analysis of 6PPD-quinone using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that extractions made with the reported ASE method demonstrated significantly higher absolute recovery (80-96%) of the extracted internal standard, [13C6]-6PPD-quinone, than sonication-based extractions (74-80%) in both fish tissue sample types. The proposed ASE method shows acceptable limits of quantification (0.37-0.67 ng g-1), linearity (R2 > 0.996), and repeatability (RSD ≤ 9%). This work advances research capabilities for investigations on the toxicokinetic processes of 6PPD-quinone in biological samples.
    Keywords:   N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine-quinone; accelerated solvent extraction; biological sample; coho salmon; liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10.1093/etojnl/vgaf151
  46. bioRxiv. 2025 Jun 01. pii: 2025.05.29.656472. [Epub ahead of print]
    Expanding the MR1 ligandome using chemical class-specific fragmentation and molecular networking.
    Chiara Lecchi, Alessandro Vacchini, Stefano Sainas, Marco L Lolli, Matthew Luedtke, Lucia Mori, Gennaro De Libero, Silvia Balbo, Peter W Villalta.
      The identification and subsequent characterization of unknown analytes using mass spectrometry presents a long-standing challenge across many research fields, particularly when analyte levels are low and the compound class is underrepresented in mass spectral databases. We have developed a data analysis workflow for investigating classes of small molecules and demonstrated its application through the reanalysis of data collected to probe for modified nucleoside MR1-presented antigens. We reanalyzed the datasets to screen for additional classes of compounds within the MR1 ligandome using Compound Discoverer, a commercial software package designed for metabolomic analysis, featuring fragmentation filtering nodes, molecular networking, and spectral database searching. Our study identified two compound classes that bind to MR1. One class includes compounds characterized by the presence of a ribityl substructure and molecular formulas consistent with structural similarity to riboflavin, where the most abundant compound differs from riboflavin by two additional oxygen atoms and one fewer carbon atom. A second class comprises an adenosine monophosphate isomer and larger analytes that are putatively identified as consisting of di- and tri-covalently bound nucleotides. The application of our analytical approach to characterize the MR1 ligandome demonstrates the power of combining compound-class fragmentation, molecular networking, and mass spectral database searching in exploring receptor ligandomes and, more generally, identifying novel classes of compounds.
    DOI:  https://doi.org/10.1101/2025.05.29.656472
  47. Methods Mol Biol. 2025 ;2925 231-239
    Separation and Quantification of Isomeric Forms of Aminobutyric Acids.
    Kamal Awad, Akhilla Kumar, Marian N Aziz, Leticia Brotto, Zhiying Wang, Lynda Bonewald, Marco Brotto.
      The biological actions of mirror-image enantiomers, such as L- and D-enantiomers of amino acids, can differ significantly due to their distinct interactions with biological systems. This is particularly evident in the case of amino acids like L- and D-BAIBA (beta-aminoisobutyric acid) and AABA (alpha-aminoisobutyric acid). These enantiomers have similar physical and chemical properties, making it challenging to distinguish and quantify them accurately using conventional methods like LC-MS/MS (liquid chromatography-tandem mass spectrometry). LC-MS/MS protocols have traditionally quantified these amino acids together in general forms. For example, BAIBA was generally detected without separating L- from D-BAIBA isoform. In this study, we introduced a novel methodology that not only overcomes these challenges but also accurately identifies isomers and enantiomers of aminobutyric acid, thereby significantly advancing the fields of analytical chemistry, biochemistry, and pharmacology.
    Keywords:  (R)-3-aminoisobutyric acid (D-BAIBA); LC-MS/MS; γ-aminobutyric acid (GABA)
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_16
  48. Anal Chem. 2025 Jun 11.
    Single-Cell Multiomic MALDI-MSI Analysis of Lipids and N-Glycans through Affinity Array Capture.
    James W Dressman, Muhammed F Bayram, Peggi M Angel, Richard R Drake, Anand S Mehta.
      This study presents a novel approach utilizing matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) for rapid single-cell analysis, enabling multiomic data acquisition from the same cell. Previously, single-cell approaches by MALDI-MSI have focused on increasing resolution to differentiate the analyte signal from adjacent cells in complex tissue or cultured cells. Laser ablation can occur on two separate cells, mixing single-cell data and requiring statistical computation to discern one cell's spectra from the other. Here, we describe a targeted single-cell approach that enables single-cell capture and sampling, assisted by micro-contact printing. We developed an AI-based application, SoloCell, to facilitate the automated selection of captured single cells within printed arrays. This method successfully captures thousands of single cells in a grid format, allowing for rapid lipid and N-glycan profiling from the same cell at a rate of 6 cells per second. This innovative technology combines the power of single-cell capture, AI automation, and imaging mass spectrometry to provide researchers with an unprecedented tool for unraveling the complexity of cellular populations.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06233
  49. J Sep Sci. 2025 Jun;48(6): e70193
    Development of the High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Creatine, L-Carnitine, and Creatinine in Tears.
    Helena Jurdáková, Michaela Šimurdová, Renáta Górová.
      The aim of this work was to develop the HPLC-MS/MS method for the determination of L-carnitine and creatine in tears, as these molecules can be used as biomarkers of several diseases and health conditions with neurological manifestation, simultaneously with the determination of creatinine as normalization factor. Tears were collected using a microtube during crying or using Schirmer strip by inserting it under the lower eyelid. Due to the low stability of creatine, only a few hours at room temperature, it is advisable to freeze the samples immediately after collection. Different methanol to water contents in the extraction solution were tested (0%-100%). Extraction recovery from Schirmer strips, matrix effect, and overall process efficiency were evaluated for 0.3 and 1 mL of extraction solution used. Finally, the extraction into 1 mL of 80% methanol containing 0.1% formic acid, and subsequent HPLC-MS/MS separation at isocratic conditions (methanol:water 5:95% with 0.1% formic acid and flow rate of 0.25 mL/min) using a Synergi 4 μ Polar-RP column (150 × 2 mm) and detection in the positive ion mode was used. The limits of quantifications for L-carnitine, creatine, and creatinine in tears were 0.5, 1.0, and 1.0 µmol/L, respectively. Intraday and interday precisions were up to 4.4% and bias did not exceed 12% for all analytes. Linearity was tested and approved in the range of 0.23-30 µmol/L for L-carnitine and 0.78-100 µmol/L for creatine and creatinine. Measured values of L-carnitine, creatine, and creatinine in real tear samples of healthy individuals (n = 7) were within the range of 1.1-8.2, 4.8-46.3, and 8-44.7 µmol/L, respectively. Obtained results were compared with published results in tears if available (L-carnitine and creatinine) or with other biological matrices. As our knowledge, this publication is the first that reports the content of creatine in tears.
    Keywords:  L‐carnitine; Schirmer strip; creatine; liquid tears; matrix effect
    DOI:  https://doi.org/10.1002/jssc.70193
  50. Anal Chim Acta. 2025 Sep 01. pii: S0003-2670(25)00637-3. [Epub ahead of print]1365 344243
    Functionalized toothpicks for solid-phase microextraction of drugs from oral fluids.
    Patricia García-Atienza, Jaime Millán-Santiago, Rafael Lucena, Soledad Cárdenas, Sergio Armenta, José Manuel Herrero-Martínez, Héctor Martínez-Pérez-Cejuela.
       BACKGROUND: Efficient extraction techniques with high selectivity and sensitivity are critical for drug analysis in biological matrices. Traditional methods often face challenges when dealing with semi-polar and ionic drugs, necessitating the development of improved sorbents. To address this issue, we propose the use of carboxymethylated wooden toothpicks (c-WTs) as a novel sorbent for solid-phase micro-extraction (SPME). This approach aims to improve drug extraction from oral fluids, offering a more effective and sustainable solution compared to conventional sorbents.
    RESULTS: Wooden toothpicks were modified through sodium chloroacetate oxidation under mild conditions, introducing carboxyl groups that enhance analyte-sorbent interactions via electrostatic forces. Characterization via infrared spectroscopy and scanning electron microscopy confirmed the successful derivatization, showing an increase in surface roughness compared to unmodified toothpicks. The optimized SPME method was applied to extract a range of 22 drugs, including amphetamines, opioids, benzodiazepines, and cannabinoids, from oral fluids. The validation of the method demonstrated excellent performance, with recovery rates ranging from 68 to 120 %. Precision was high, with relative standard deviations (RSD) below 10 %. The method also exhibited low limits of quantification (1-5 μg L-1) when analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), indicating its potential for reliable and sensitive drug analysis in biological matrices.
    SIGNIFICANCE: This study presents an innovative, sustainable approach for drug extraction using natural-based sorbents. The use of c-WTs offers a cost-effective, selective, and environmentally friendly alternative to traditional methods. This work contributes to the development of efficient SPME techniques for complex biological samples, advancing analytical methodologies in forensic and clinical applications.
    Keywords:  Biological samples; Drugs; Solid-phase microextraction; Sorbent; Wooden toothpicks
    DOI:  https://doi.org/10.1016/j.aca.2025.344243
  51. Anal Chem. 2025 Jun 12.
    On Selecting Robust Approaches for Learning Predictive Biomarkers in Metabolomics Data Sets.
    Thibaud Godon, Pier-Luc Plante, Jacques Corbeil, Pascal Germain, Alexandre Drouin.
      Metabolomics, the study of small molecules within biological systems, offers insights into metabolic processes and, consequently, holds great promise for advancing health outcomes. Biomarker discovery in metabolomics represents a significant challenge, notably due to the high dimensionality of the data. Recent work has addressed this problem by analyzing the most important variables in machine learning models. Unfortunately, this approach relies on prior hypotheses about the structure of the data and may overlook simple patterns. To assess the true usefulness of machine learning methods, we evaluate them on a collection of 835 metabolomics data sets. This effort provides valuable insights for metabolomics researchers regarding where and when to use machine learning. It also establishes a benchmark for the evaluation of future methods. Nonetheless, the results emphasize the high diversity of data sets in metabolomics and the complexity of finding biologically relevant biomarkers. As a result, we propose a novel approach applicable across all data sets, offering guidance for future analyses. This method involves directly comparing univariate and multivariate models. We demonstrate through selected examples how this approach can guide data analysis across diverse data set structures, representative of the observed variability. Code and data are available for research purposes.
    DOI:  https://doi.org/10.1021/acs.analchem.5c01049
  52. Methods Mol Biol. 2025 ;2925 329-382
    Protocols for Metabolomic Analyses to Assess Changes in Central Carbon Metabolism from Neural Tissue.
    Mai Noureldein, Darcy Cochran, Fatema Bhinderwala, Shulei Lei, Jade Woods, Jordan Rose, Darrell D Marshall, Eli Riekeberg, Aline De Lima Leite, Martha Morton, Eric D Dodds, Rodrigo Franco, Robert Powers.
      Metabolomics has been successfully applied to study neurological and neurodegenerative disorders, including Parkinson's disease for the following purposes: (1) identifying potential biomarkers of onset and disease progression; (2) identifying novel mechanisms of disease progression; and (3) assessing treatment prognosis and outcome. Reproducible and efficient extraction of metabolites is imperative to the success of any metabolomics investigation. Unlike other OMICS techniques, the composition of the metabolome can be negatively impacted by the preparation, processing, and handling of these samples. The proper choice of data collection, preprocessing, and processing protocols are similarly important to the design of an effective metabolomics experiment. Likewise, correctly applying univariate and multivariate statistical methods is essential for providing biologically relevant insights. In this chapter, we have outlined a detailed metabolomics workflow that addresses these issues. A step-by-step protocol from preparing neuronal cells and metabolomic tissue samples to their metabolic analyses using nuclear magnetic resonance, mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and chemometrics is presented.
    Keywords:  Chemometrics; MALDI imaging; Mass Spectrometry; Metabolomics; NMR; Neurodegeneration; Parkinson’s disease
    DOI:  https://doi.org/10.1007/978-1-0716-4534-5_24
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