bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–11–30
forty-four papers selected by
Sofia Costa, Matterworks
  1. Comprehensive Quantitative Profiling of Less Polar Lipids in Human Plasma Using Validated Reversed-Phase UHPSFC/MS/MS.
  2. Chiral High-Performance Liquid Chromatography (HPLC) in Targeted Metabolomics.
  3. Expanding Metabolome Coverage in LC-MS/MS Analysis through Hydralazine-Based Multifunctional Derivatization.
  4. Multiclass Assays for Measuring Environmental Chemical Mixture Exposure: Analytical Methodologies and Applications in Exposomics Research.
  5. Two-Dimensional LC-MS/MS Determination of Chiral Amino Acids in Real-World Samples.
  6. SMAnalyst: A Web Server for Spatial Metabolomic Data Analysis and Annotation.
  7. An integrated LC-MS approach to the metabolome profile in the whole-body tissue of adult zebrafish.
  8. Development of a hydrophilic interaction-based procedure for the extraction, separation, and determination of therapeutic oligonucleotides.
  9. Rapid and Sensitive Quantitation of Fipronil and Its Metabolites Fipronil Sulfone, Fipronil Sulfide, and Fipronil Desulfinyl in Zebrafish Tissues by UHPLC-MS/MS.
  10. A novel Transformer-MLP fusion network for metabolite identification from mass spectra.
  11. An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of dehydroepiandrosterone sulfate in human serum and plasma.
  12. Simultaneous Determination of Olmesartan and Hydrochlorothiazide in Human Plasma by UPLC-MS/MS Method and Its Application to a Bioequivalence Study.
  13. Simulating Tandem Mass Spectra for Small Molecules using a General-Purpose Large-Language Model.
  14. Negative Paper Spray Ionization Mass Spectrometry for the Determination of Endocrine-Disrupting Chemicals with Application to Paraben Analysis in Cosmetics.
  15. Quantification of esterified oxylipins following HILIC-fractionation of lipid classes.
  16. Laser Desorption-Rapid Evaporative Ionization Mass Spectrometry (LD-REIMS): A New Tool for the High-Throughput Metabolomic and Lipidomic Profiling of Live Cells.
  17. Liquid chromatography-mass spectrometry quantitation and prevalence of medetomidine and xylazine in New Haven, Connecticut.
  18. RSR-MSI: Reference-Based Super-Resolution for Mass Spectrometry Imaging of Tissues and Single Cells.
  19. LC-MS/MS Detection of Tryptophan, Kynurenine, Kynurenic Acid, and Quinolinic Acid in Urine Samples from Drug-Positive and Illicit Drug-Negative Patients with a Known History of Substance Use Disorder.
  20. AI redefines mass spectrometry chemicals identification: retention time prediction in metabolomics and for a Human Exposome Project.
  21. Development and validation of an LC-MS/MS method for the simultaneous detection of urinary inflammatory biomarkers in a Flemish birth cohort.
  22. Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Quantification of 3,14,19-Triacetyl Andrographolide in Rat Plasma: Application to Preclinical Pharmacokinetic Studies.
  23. LC-MS/MS quantitation of the primary reduced metabolites of progesterone in serum during the third trimester of human pregnancy reveals a potential role for 20β-hydroxyprogesterone and 5β-dihydroprogesterone in functional progesterone withdrawal.
  24. High-Throughput Mass Spectral Library Searching of Small Molecules in R with NIST MSPepSearch.
  25. A widely targeted analytical method for oxylipins including enantiomers using chiral liquid chromatography tandem mass spectrometry.
  26. Analysis of the Isomers of New Chiral Psychoactive Substances Using Enantioselective High-Performance Liquid Chromatography-Mass Spectrometry.
  27. Learning from All Views: A Multiview Contrastive Framework for Metabolite Annotation.
  28. Liver microsomal fraction induced small molecule isotope labeling via catalyzed H/D and 16O/18O isotope exchange reactions: A novel approach for obtaining isotope-labeled compounds.
  29. Simultaneous Quantification of Multiple Analytes in Rat Plasma by UHPLC-MS/MS Following Oral Administration of Gastrodiae Rhizoma Extract for Pharmacokinetic Evaluation.
  30. Storing Mass-Spectrometry Data in Simple Databases Enables Flexible and Intuitive Exploration without Time or Space Penalties.
  31. Quantification of a sea lamprey (Petromyzon marinus) pheromone antagonist in river water using ion pairing solid phase extraction coupled with liquid chromatography-tandem mass spectrometry.
  32. Current trends in liquid chromatography-mass spectrometry analysis of small interfering RNAs: A short review.
  33. Targeted Metabolomic Strategy for Epigenetic Modification-Related Metabolites Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.
  34. Dual one-step LC-MS/MS methods for hair analysis in drug-facilitated crime: application to an alleged Fraud-Related case.
  35. Mass Spectrometry-Based Applications in Tannin Analytics: From Qualitative and Quantitative Analyses to Biological Activity.
  36. A Novel and Validated GC-MS/MS Method for the Detection of Four Opioids and Seven Fentanoids in Oral Fluid for Forensic Applications.
  37. Metabolomic and lipidomic atlas of human hair across its length.
  38. Metabolomics and Pharmacometabolomics: Advancing Precision Medicine in Drug Discovery and Development.
  39. Green and Emerging Microextraction Strategies for Bioanalytical Determination of Hormones: Trends, Challenges, and Applications.
  40. Simultaneous determination of iloperidone and its metabolites in rat plasma using a novel UPLC-MS/MS method: an application for drug-drug interaction.
  41. Tunable dual-ligand ZIF-8-90 effervescent tablet-assisted dispersive solid phase extraction for monitoring of both PAHs and their hydroxyl metabolites: from external to internal exposure analysis.
  42. Multidimensional Mass Spectral Similarity Algorithm: Discriminate Disaccharide and Flavonoid Isomers Coupled with Online Energy-Resolved Acquisition and Electron Activation Dissociation.
  43. Optimisation of a One-Step Reusable Immuno-Affinity Purification Method for the Analysis and Detection of Fumonisin Mycotoxins in Foods and Feeds.
  44. Lipidome mapping of natural rubber: Detailed investigation into the influence of geographical origin and treatment using liquid chromatography and high-resolution tandem mass spectrometry.
  1. Anal Chem. 2025 Nov 23.
    Comprehensive Quantitative Profiling of Less Polar Lipids in Human Plasma Using Validated Reversed-Phase UHPSFC/MS/MS.
    Zuzana Lásko, Veronika Šubrtová, Ondřej Peterka, Robert Jirásko, Michal Holčapek.
      The quantitative analysis of lipids is challenging due to their structural diversity and the coexistence of numerous isomers in biological samples. Here, we present a robust and validated method based on reversed-phase ultrahigh-performance supercritical fluid chromatography-tandem mass spectrometry (RP-UHPSFC/MS/MS) for the high-throughput profiling of less polar lipids in human plasma. The sample preparation workflow involves an initial Folch-based extraction, followed by chemical derivatization with benzoyl chloride, and the final liquid-liquid extraction into hexane to isolate less polar acylated analytes. This strategy enables the sensitive profiling of six major lipid classes (TG, DG, MG, SE, ST, and FA) in positive ion mode in less than 18 min. The use of two C18 columns in series provides high chromatographic resolution, sufficient to separate isomeric species, including cis/trans and positional isomers of double bonds in fatty acids. The method was rigorously validated for five lipid classes, except free FA, and the implementation of response factors was shown to be essential for the accurate quantification of sterol esters. A total of 147 lipid species were quantified in NIST SRM 1950 human plasma, demonstrating its suitability for large-scale lipidomic studies focused on less polar lipids.
    DOI:  https://doi.org/10.1021/acs.analchem.5c04668
  2. Methods Mol Biol. 2026 ;2994 197-229
    Chiral High-Performance Liquid Chromatography (HPLC) in Targeted Metabolomics.
    Xiaoqing Fu, Michael Lämmerhofer.
      Enzymatic reactions usually occur with a high substrate, product, or substrate-product stereoselectivity. In many instances, information on the stereochemical disposition of biologically active metabolites is indispensable for a complete understanding of biological processes and metabolic states. This raised interest in structural isomerism and stereoisomerism in metabolomics and lipidomics. While structural and geometrical isomers, as well as diastereomers, can be principally distinguished by achiral liquid chromatography (LC) and also ion-mobility mass spectrometry (MS), enantiomers require enantioselective separation methods. While untargeted enantioselective metabolomics is mostly based on the derivatization of metabolites with a chiral derivatizing agent, followed by LC separation on achiral columns and hyphenation with high-resolution mass spectrometry, targeted enantioselective LC with chiral stationary phases mostly employs robust triple quadrupole (QqQ) mass spectrometry for detection. It is a powerful technology, yet it focuses primarily on a broad, metabolite class-wide coverage. This chapter describes four distinct fields of application of targeted enantioselective LC-tandem mass spectrometry assays for important and representative metabolite classes. The first one deals with the enantioselective analysis of oxylipins using polysaccharide-based chiral columns, with protocols applicable for platelets and plasma; the second focuses on 3-hydroxy fatty acid enantiomer separations in plasma and platelets; the third application provides two workflows for proteinogenic enantioselective amino acid analysis; and the fourth application proposes methodologies for enantioselective short branched-chain fatty acid analysis. Critical aspects are discussed.
    Keywords:  3-Hydroxy fatty acid; Amino acid; Branched alkanoic acids; Chiral derivatizing agent; Chiral stationary phase; LC–MS; Lipidomics; Mass spectrometry; Metabolite; Metabolomics; Oxylipin
    DOI:  https://doi.org/10.1007/978-1-0716-5023-3_11
  3. Anal Chem. 2025 Nov 24.
    Expanding Metabolome Coverage in LC-MS/MS Analysis through Hydralazine-Based Multifunctional Derivatization.
    Zhiye Yan, Weiwei Tang, Bin Li.
      Metabolomics is a potent tool used for discovering disease biomarkers and investigating intricate mechanisms of diseases by analyzing metabolite changes in biological systems. However, challenges in metabolite detection have not been fully addressed due to their vast physicochemical diversity and broad concentration range, impacting comprehensive profiling of the metabolome, particularly for certain metabolites with limited detection sensitivity. Therefore, a novel chemical derivatization method based on hydralazine (HZN) was developed for highly sensitive detection of various poorly ionized small metabolites in biological samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). HZN-based multifunctional derivatization mainly targeted metabolites with carbonyl, carboxyl, and phosphoryl groups, such as free fatty acids, keto-acids, hydroxy acids, bile acids, fatty aldehydes, and phosphoryl compounds. Main parameters affecting the derivatization efficiency were investigated, such as the condensation reagent, and optimal conditions were obtained. The labeling of HZN enabled the generation of characteristic fragment ions through collision-induced dissociation, avoiding the laboriousness of deducing MS/MS fragmentation and facilitating large-scale quantitative analysis. The sensitivities of HZN-derivatized metabolites improved significantly, ranging from 10 to 5000-fold, and the limits of quantitation for derivatized metabolites varied from 5 pM to 100 nM, depending on their structures. Furthermore, alterations of various gut microbiota-derived metabolites in mice fed with a high-fat diet were unraveled using the HZN-based derivatization method, indicating its promising applications in the investigation of disease-related changes in the metabolome.
    DOI:  https://doi.org/10.1021/acs.analchem.5c02101
  4. Metabolites. 2025 Nov 16. pii: 742. [Epub ahead of print]15(11):
    Multiclass Assays for Measuring Environmental Chemical Mixture Exposure: Analytical Methodologies and Applications in Exposomics Research.
    Ravikumar Jagani, Jasmin Chovatiya, Divya Pulivarthi, Anil K Meher, Dhavalkumar Patel, Hiraj Patel, Sandipkumar Teraiya, Syam S Andra.
       BACKGROUND/OBJECTIVES: The exposome includes all environmental exposures throughout a lifetime and profoundly influences health and disease, reflecting the totality of environmental chemical exposures throughout an individual's life, encompassing both natural and anthropogenic chemicals from external sources. Conventional methods for environmental chemical analysis have generally concentrated on individual representatives or substance classes; however, single analyte/class techniques are impractical for extensive epidemiological studies that require the analysis of thousands of samples, as anticipated for forthcoming exposome-wide association studies. This narrative review analyzes the evolution and implementation of multiclass assays for measuring ambient chemical exposure, emphasizing analytical techniques that provide the concurrent quantification of various chemical classes.
    METHODS: This narrative review consolidates existing literature on multiclass analytical methodologies for measuring exposure to environmental chemical mixtures, encompassing mass spectrometry platforms, sample preparation techniques, chromatographic separation methods, and validation strategies for thorough exposure assessment in human biomonitoring research. The review includes liquid chromatography-mass spectrometry techniques, solid-phase extraction methods, and data analysis strategies for both targeted and non-targeted study.
    RESULTS: Multi-class methodologies provide the concurrent quantification of compounds from many classes without the necessity for distinct conventional procedures, thus minimizing time, expense, and sample volume. The robustness of the method indicates appropriate extraction recovery and matrix effects between 60 and 130%, inter-/intra-day precision under 30%, and remarkable sensitivity with detection limits from 0.015 to 50 pg/mL for 60-80% of analytes in the examined human matrices. The methodology facilitates the concurrent identification of the endogenous metabolome, food-associated metabolites, medicines, home chemicals, environmental contaminants, and microbiota derivatives, including over 1000 chemicals and metabolites in total.
    CONCLUSIONS: These thorough analytical methods deliver the requisite performance for extensive exposome-wide association studies, yielding quantitative results and uncovering unforeseen exposures, thereby augmenting our comprehension of the chemical exposome, which is essential for advancing disease prevention in public health and personalized medicine.
    Keywords:  LC-HRMS; LC-MS/MS; biomonitoring; chemical exposome; environmental contaminants; exposome-wide association studies; matrix effects; multiclass assays; multiple reaction monitoring; solid phase extraction
    DOI:  https://doi.org/10.3390/metabo15110742
  5. Methods Mol Biol. 2026 ;2994 231-244
    Two-Dimensional LC-MS/MS Determination of Chiral Amino Acids in Real-World Samples.
    Chiharu Ishii, Kenji Hamase.
      Chiral amino acid analysis is currently gathering attention since several D-form amino acids (minor enantiomers of the predominantly observed L-amino acids) have been demonstrated to be the new physiologically active substances and biomarkers. However, the amounts of these D-amino acids in biological samples are trace in most cases, and sensitive and selective analytical methods are essential for their determination. In the present chapter, multidimensional liquid chromatography (LC) systems, especially the design and development of two-dimensional LC-tandem mass spectrometry (MS/MS) systems, are summarized, and the applications of the methods to the determination of chiral amino acids in various real-world matrices, including human urine, plasma, fermented products, and protein samples, are described.
    Keywords:  Amino acids; Chiral separation; MS/MS; Two-dimensional LC
    DOI:  https://doi.org/10.1007/978-1-0716-5023-3_12
  6. Biomolecules. 2025 Nov 06. pii: 1562. [Epub ahead of print]15(11):
    SMAnalyst: A Web Server for Spatial Metabolomic Data Analysis and Annotation.
    Zhanlong Mei, Xiaolian Ning, Haoke Deng, Lingyun Chen, Yun Zhao, Jin Zi.
      Spatial metabolomics is a rapidly advancing field offering powerful insights into metabolic heterogeneity in biological tissues. However, its widespread adoption is hindered by fragmented tools and the lack of comprehensive, open-source GUI software covering the full analytical workflow (quality control, preprocessing, identification, pattern, and differential analysis). To address this, we developed SMAnalyst, an open-source, integrated web-based platform. SMAnalyst consolidates core functionalities, including multi-dimensional data quality assessment (background consistency, intensity, missing values), a comprehensive metabolite annotation scoring system (mass accuracy, isotopic similarity, adduct evidence), and dual-dimension spatial pattern discovery (metabolite co-expression and pixel clustering). It also offers flexible differential analysis (cluster- or user-defined regions). With its intuitive GUI and modular workflow, SMAnalyst significantly lowers the analysis barrier, by providing a unified solution that eliminates the need for tool switching and advanced computational skills. Tested with a mouse brain dataset, SMAnalyst efficiently handles large-scale data (e.g., >14,000 pixels, >3000 ion peaks), effectively filling a critical gap in integrated analytical solutions for spatial metabolomics.
    Keywords:  differential analysis; metabolite annotation; quality control; spatial metabolomics; spatial pattern discovery; web-based platform
    DOI:  https://doi.org/10.3390/biom15111562
  7. Anal Methods. 2025 Nov 24.
    An integrated LC-MS approach to the metabolome profile in the whole-body tissue of adult zebrafish.
    Mengmeng Sun, Qiyu Yao, Linjiang Wei, Zhi Zhou.
      Zebrafish (Danio rerio) has served as a crucial experimental model in biomedical and environmental research. Compared to the more commonly used embryonic and juvenile stages, adult zebrafish possesses a complete inter-organ metabolic network, which is advantageous in the development of a chronic disease model. However, there are very few comprehensive studies on metabolomics methods for adult zebrafish. Herein, we have implemented an integrated approach that combines hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) to maximize metabolite detection and optimized key parameters in sample pretreatment, including tissue homogenization, extraction solvents, the extraction solvent addition method, and re-dissolution solvents. As such, we have established an untargeted metabolomics analysis method, utilizing ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) technology, which is suitable for whole-tissue analysis. This method has demonstrated satisfactory precision, accuracy, linearity, and sample stability in methodology validation tests. Through metabolite database retrieval using custom scripts, a total of 620 metabolites were annotated, highlighting the complementarity of RPLC and HILIC, as well as the method's extensive coverage. Its applicability was verified through a 20 days observation period experiment, screening out 110 differential variables associated with zebrafish growth. Of these, 23 are known compounds that are primarily related to glycerophospholipid metabolism, phenylalanine, tyrosine and tryptophan biosyntheses, and phenylalanine metabolism. This study has clarified the experimental conditions for metabolomics studies based on adult zebrafish, providing novel methodological approaches for related disease or environmental exposure research.
    DOI:  https://doi.org/10.1039/d5ay01240f
  8. Anal Chim Acta. 2026 Jan 01. pii: S0003-2670(25)01202-4. [Epub ahead of print]1381 344808
    Development of a hydrophilic interaction-based procedure for the extraction, separation, and determination of therapeutic oligonucleotides.
    Zuzana Vosáhlová, Szymon Bocian, Sylwia Studzińska.
       BACKGROUND: For analyzing oligonucleotide impurities and metabolites in serum samples, where mass spectrometry detection is essential, hydrophilic interaction liquid chromatography utilizing fully mass spectrometry compatible mobile phases is the best choice. However, reliable analysis must be preceded by a reliable extraction method providing sufficient and repeatable recoveries. Thus, this work aimed to develop a simple and repeatable methodology for extracting and analyzing modified oligonucleotides differing in length (4mer-14mer), both under hydrophilic interaction conditions.
    RESULTS: A comprehensive retention study examining the effects of mobile phase composition and temperature on retention and peak shape was conducted for the zwitterionic and amide columns. Based on the results, the amide column was chosen for further study, mainly because the zwitterionic column does not permit elution of longer oligonucleotides (>10mer). In addition, the amide column exhibits greater robustness (minor effects of tested parameters on chromatographic performance), which ensures higher repeatability. The optimization of mass spectrometry parameters was performed to obtain the highest possible sensitivity, which is well well-known issue because of adducts formation. A validated procedure was applied to analyze enriched serum extracts - a newly synthesized amide-based sorbent was used for the dispersive solid-phase extraction. The conditions for adsorption and desorption, which are driven by a weak ion-exchange mechanism and thus by pH change, were optimized to obtain the highest recoveries.
    SIGNIFICANCE: A fully hydrophilic interaction-based procedure was utilized for the first time f extraction and analysis of a mixture of 5 modified oligonucleotides differing in length from enriched serum. This method proved to be simple, repeatable, and provides recoveries higher than 75 % (RSD <5 %).
    Keywords:  Amide adsorbent; Dispersive solid phase extraction; Hydrophilic interaction liquid chromatography; Mass spectrometry; Modified antisense oligonucleotides
    DOI:  https://doi.org/10.1016/j.aca.2025.344808
  9. J Sep Sci. 2025 Dec;48(12): e70326
    Rapid and Sensitive Quantitation of Fipronil and Its Metabolites Fipronil Sulfone, Fipronil Sulfide, and Fipronil Desulfinyl in Zebrafish Tissues by UHPLC-MS/MS.
    Meichen Liu, Yingxia Guo, Jinhe Liu, Zihan Tang, Yufeng Guo, Xintao Liu, Deshan Zhou, Lei Yin, Meiyun Shi.
      An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous determination of fipronil and its three metabolites (fipronil sulfone, fipronil sulfide, and fipronil desulfinyl) in zebrafish tissues. Protein precipitation was employed for sample preparation, followed by chromatographic separation on a C18 column. The method demonstrated excellent linearity (r2 > 0.99) across a range of 0.1-10 ng/mL, with a lower limit of quantification (LLOQ) of 0.1 ng/mL for all analytes. Validation results confirmed acceptable accuracy (-5.0% to 8.9%), precision (0.6%-13.2%), recoveries (86.3%-113.6%), and minimal matrix effects (86.0%-110.6%). When applied to zebrafish exposed to 50 µg/L fipronil for 12 h, the method revealed preferential accumulation of fipronil and fipronil sulfone in liver tissues, while lower levels were detected in muscle tissues. Fipronil sulfide was rarely detected, and fipronil desulfinyl concentrations were below the LLOQ (0.1 ng/mL) in all tested samples. This reliable and sensitive method is well-suited for environmental toxicology studies, pesticide residue monitoring, and ecological risk assessment.
    Keywords:  UHPLC‐MS/MS; fipronil; metabolites; tissue distribution study; zebrafish
    DOI:  https://doi.org/10.1002/jssc.70326
  10. Talanta. 2025 Nov 17. pii: S0039-9140(25)01614-5. [Epub ahead of print]299 129123
    A novel Transformer-MLP fusion network for metabolite identification from mass spectra.
    Xiaofeng Zhang, Ming Yan, Yian Liu, Lingyun Xue, Anqi Chen, Shundi Hu, Luhong Wen, Ping Xu.
      Accurate metabolite identification remains a major challenge in untargeted metabolomics, particularly for novel compounds that are absent from existing spectral libraries. In this work, we propose a novel Feature Fusion Network (FFNet) based on Transformer and multilayer perceptron (MLP), a dual-stream architecture specifically designed to predict molecular fingerprints from mass spectra. FFNet incorporates a transformer-based pathway that captures the global context of spectral data and an MLP-based pathway that focuses on extracting local spectral features. These complementary representations are integrated through an attentive fusion mechanism to produce comprehensive molecular fingerprints. Extensive evaluations on the General Metabolite Identification Set (GMIS, 17,267 spectra), a custom-built test set designed for benchmarking metabolite identification, and the MassBank of North America (MoNA, 1243 spectra) dataset demonstrate that FFNet consistently outperforms baseline neural network models in both fingerprint prediction and metabolite identification tasks. Moreover, in structure elucidation experiments using the CASMI 2022 dataset, FFNet effectively retrieves candidate molecules with high structural similarity to unknown compounds, even in the absence of exact matches within the spectral library. These findings suggest that neural feature fusion significantly improves mass spectral analysis and enables more reliable metabolite identification in complex biological samples.
    Keywords:  Deep learning; Mass spectrometry; Metabolite identification; Metabolomics; Molecular fingerprinting
    DOI:  https://doi.org/10.1016/j.talanta.2025.129123
  11. Clin Chem Lab Med. 2025 Dec 01.
    An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of dehydroepiandrosterone sulfate in human serum and plasma.
    Sara Cheikh Ibrahim, Neeraj Singh, Katrin Gradl, Friederike Bauland, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Christian Geletneky, Judith Taibon.
       OBJECTIVES: Dehydroepiandrosterone sulfate (DHEAS), the sulfate ester of dehydroepiandrosterone, is one of the most common steroid hormones in the human body and the precursor of several other androgens. It is primarily used as a diagnostic or prognostic indicator in adrenal and reproductive disorders. Present immunoassays for DHEAS lack sensitivity and specificity, being vulnerable to cross-reactivity with endogenous interferences. Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed to quantify DHEAS in human serum/plasma.
    METHODS: We ensured traceability to the International System of Units by using quantitative nuclear magnetic resonance to characterize a commercially available DHEAS reference material used for assay calibration. To mitigate matrix effects and prevent interference co-elution, a two-dimensional heart-cut LC method was employed for LC-MS/MS, in combination with a solid phase extraction sample preparation protocol. Selectivity was determined by spiking the prepared internal standard with the interferences testosterone, epi-testosterone, dehydroepiandrosterone, 5α-dihydrotestosterone, and estrone, in analyte free matrix. A post-column infusion experiment and comparison of standard line slopes were performed to evaluate matrix effects. Precision and accuracy were assessed via a multi-day validation experiment, and variability components estimated using analysis of variance-based variance-components analysis. Measurement uncertainty (MU) was evaluated in compliance with current guidelines.
    RESULTS: This RMP was suitable for analyzing DHEAS within the 0.800 to 8,400 ng/mL (2.17-22,800 nmol/L) range, demonstrating selectivity, sensitivity, and matrix-independence. Trueness and accuracy assessment revealed a relative bias (n=6) between -1.9 and 0.3 % for surrogate matrix samples (except for 5.9 % at the lowest level), -2.3 to 3.6 % for Li-heparin plasma samples and sample dilutions, and an overall bias between 0.7 and 1.8 % (n=60), indicating no statistically significant bias. The measurement process resulted in standard measurement uncertainties (MUs) ranging from 4.0 to 5.6 % for the low range and 3.5-4.2 % for the high range. At a 95 % confidence level (k=2), these uncertainties expanded to 7.9-11.1 % and 7.1-8.3 %, respectively. Reference values, determined from six measurements over multiple days (n=6), had standard MUs between 1.6 and 2.1 % for the low range and 0.9-1.7 % for the high range, with expanded MUs of 3.2-4.3 % and 1.9-3.5 %.
    CONCLUSIONS: This RMP exhibited high analytical performance for DHEAS quantification and met requirements for measurement uncertainty. Additionally, it enabled differentiation between the DHEAS and other androgens. Consequently, this RMP is suitable for routine assay standardization and clinical sample evaluation.
    Keywords:  SI units; dehydroepiandrosterone sulfate; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability
    DOI:  https://doi.org/10.1515/cclm-2025-0179
  12. Biomed Chromatogr. 2026 Jan;40(1): e70254
    Simultaneous Determination of Olmesartan and Hydrochlorothiazide in Human Plasma by UPLC-MS/MS Method and Its Application to a Bioequivalence Study.
    Jie Shen, Lin Zhang, Jianbang Wu, Yan Zhou, Minhui Wang, Changmao Wang, LingLing Zhu, Yaqin Wang, Ping Wu, Xiaocui Xia, Wanyu Wang, Yamin Liu, Yuanwei Jia.
      A simple, sensitive and efficient ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma has been developed and validated. Using olmesartan-d4 and hydrochlorothiazide-13C-d2 as stable isotope-labeled internal standard (SIL-IS). Plasma samples were processed by protein precipitation. Separated on a ZORBAX Eclipse XDB-Phenyl column (150 mm × 4.6 mm, 5 μm) using a gradient elution with mobile phases methanol and 5 mM ammonium acetate. Detected by electrospray ionization (ESI) in negative ion mode of multiple reaction monitoring (MRM), mass transition ion pairs were m/z 445.1 → 149.0 for olmesartan, m/z 295.9 → 204.9 for hydrochlorothiazide, m/z 449.1 → 149.1 for olmesartan-d4, and m/z 301.0 → 272.0 for hydrochlorothiazide-13C-d2. Linear ranges were 10-2000 ng/mL for olmesartan and 1.5-300 ng/mL for hydrochlorothiazide. Mean recoveries of olmesartan, hydrochlorothiazide, olmesartan-d4, and hydrochlorothiazide-13C-d2 were 91.31%, 99.34%, 92.62%, and 98.61%, respectively. Our method was well validated in selectivity, carryover, lower limit of quantification (LLOQ), calibration curve, accuracy, precision, dilution effect, matrix effect (normal, hyperlipidemic, and hemolyzed matrices), stability, recovery, and IRS. It was successfully applied in a bioequivalence study of olmesartan medoxomil and hydrochlorothiazide tablets (20/12.5 mg) in healthy Chinese volunteers.
    Keywords:  UPLC–MS/MS; hydrochlorothiazide; olmesartan; pharmacokinetic; plasms
    DOI:  https://doi.org/10.1002/bmc.70254
  13. bioRxiv. 2025 Nov 11. pii: 2025.11.10.687298. [Epub ahead of print]
    Simulating Tandem Mass Spectra for Small Molecules using a General-Purpose Large-Language Model.
    Tuan Nguyen, Dinesh Barupal.
      We show a practical application of the Google Gemini large-language-model for simulating tandem mass spectra for compounds from the Blood Exposome Database. This approach bypasses the need for domain-specific model training, suggesting that the chemical fragmentation knowledge could be latently encoded within the Gemini model. General-purpose LLMs represent a useful and accessible tool for expanding in-silico spectral libraries and may accelerate the compound annotation in mass spectrometry-based metabolomics and exposomics.
    DOI:  https://doi.org/10.1101/2025.11.10.687298
  14. Molecules. 2025 Nov 10. pii: 4356. [Epub ahead of print]30(22):
    Negative Paper Spray Ionization Mass Spectrometry for the Determination of Endocrine-Disrupting Chemicals with Application to Paraben Analysis in Cosmetics.
    Seonyoung Cho, Sarmila Shrestha Amatya, Hyerin Bahng, Eungyeong Lee, Yunsang Ko, Sangwon Cha.
      Paper spray ionization mass spectrometry (PSI-MS) enables rapid analysis with minimal sample preparation, yet negative-ion mode performance has been limited by poor sensitivity and unstable signals, similar to conventional electrospray ionization. In this study, we optimized negative PSI tandem MS (MS/MS) for twelve endocrine-disrupting chemicals (EDCs) and related biomarkers-including bisphenols, phthalates, parabens, and substituted phenols-used as model analytes. A systematic solvent and additive screen identified 1 mM ammonium fluoride in methanol and 0.1% ammonium hydroxide in 9:1 MeOH/carbon tetrachloride as optimal conditions, providing enhanced deprotonated-ion intensities and improved stability. Calibration curves generated under these conditions showed excellent linearity, with limits of quantitation (LOQs) in the low-ppb range. Application to cosmetic formulations demonstrated reliable paraben quantitation. In fortified hand cream, LOQs below 1 mg/kg were achieved, with recoveries of 93-110% and intra- and inter-day precision below 10% RSD. Notably, PSI-MS/MS performance was comparable to LC-MS/MS, without a separation step. These results demonstrate the feasibility of optimized negative PSI-MS as a sensitive and robust tool for paraben determination in cosmetics and highlight its potential as a versatile platform for broader EDC quantification.
    Keywords:  cosmetics; endocrine-disrupting chemicals; paper spray ionization; paraben; tandem mass spectrometry
    DOI:  https://doi.org/10.3390/molecules30224356
  15. J Lipid Res. 2025 Nov 21. pii: S0022-2275(25)00213-5. [Epub ahead of print] 100950
    Quantification of esterified oxylipins following HILIC-fractionation of lipid classes.
    Luca M Wende, Laura Carpanedo, Lilli Scholz, Nadja Kampschulte, Annette L West, Philip C Calder, Nils Helge Schebb.
      Several oxylipins are lipid mediators derived from the oxidation of polyunsaturated fatty acids (PUFAs). The majority of oxylipins in biological samples occurs esterified in neutral lipids (nLs) and phospholipids (PLs). They are commonly quantified indirectly following alkaline hydrolysis providing excellent sensitivity but the information in which lipid classes the oxylipins occurred in is lost. The direct analysis of oxidized lipids is currently not sensitive enough to detect all esterified oxylipins. Here, a new hydrophilic interaction liquid chromatography (HILIC) based lipid class fractionation using solid-phase extraction (SPE) cartridges was developed separating lipids into nLs and 4 PL fractions using a single column. Esterified oxylipins in the fractions were quantified following alkaline hydrolysis to sensitively pinpoint in which lipid classes they are bound in plasma. The fractionation was extensively characterized for different lipid extracts demonstrating high separation efficiency and recovery using labeled standards and untargeted analysis of endogenous lipids. Esterified oxylipins in the fractions were quantitatively detected. Based on the results from two independent human plasma pools including SRM1950 it is shown that: hydroxy-linoleic acid- and hydroxy-α-linolenic acid-derived oxylipins are preferably bound to nLs whereas long chain hydroxy-PUFAs and PUFAs (i.e. ARA EPA and DHA) are predominantly esterified to phospholipid classes. Supplementation of n3-PUFAs for 12 months led to an increase in EPA- and -DHA-derived oxylipins in all lipid fractions with the highest increase of hydroxy-PUFAs in nLs. This demonstrates a precursor PUFA-dependent binding of oxylipins and a direct effect of diet on esterified oxylipins in plasma.
    Keywords:  esterified oxylipins; fish oil; glycerophospholipids; human plasma; hydrophilic interaction liquid chromatography; lipidomics; nutrition; omega-3 fatty acids; oxidized lipids; solid-phase extraction
    DOI:  https://doi.org/10.1016/j.jlr.2025.100950
  16. Anal Chem. 2025 Nov 27.
    Laser Desorption-Rapid Evaporative Ionization Mass Spectrometry (LD-REIMS): A New Tool for the High-Throughput Metabolomic and Lipidomic Profiling of Live Cells.
    Stefania Maneta-Stavrakaki, Aurelien Tripp, Daniel Simon, Yuchen Xiang, Adrienn Molnar, Athanasios Tsalikis, Efstathios Andreas Elia, Josephine Bunch, Julia Balog, George Poulogiannis, Zoltan Takats.
      Understanding the dynamic cellular metabolism is essential for gaining deeper insights into inter- and intracellular functions. In recent years, mass spectrometry (MS) has become the technology of choice for the biochemical characterization and profiling of cell lines, particularly when coupled with separation techniques such as liquid chromatography (LC-MS). However, these methods typically involve extensive sample preparation with potent organic solvents, which is labor-intensive, time-consuming, and incompatible with direct analysis of intact, live cells. Here, we propose the use of the ambient ionization technique Laser Desorption-Rapid Evaporative Ionization Mass Spectrometry (LD-REIMS) incorporated in an automated platform, for the high-throughput profiling of live or frozen cell monolayers, with minimal pretreatment. Validation experiments using 10 breast and colorectal cancer cell lines confirmed high accuracy, repeatability, and molecular coverage of the method, with over 400 metabolites and lipids detected and identified, including saccharides, amino acids, fatty acids, and glycerophospholipids. Of these, 144 were further confirmed and quantified with LC-MS/MS and standard compounds. We also applied the method to establish lipidomic differences across the isogenic MCF10A cells harboring either WT or MUT PIK3CA. Finally, we conducted time-series experiments on hypoxic cells, which revealed significant dynamic changes in metabolism, including lactate accumulation due to anaerobic glycolysis.
    DOI:  https://doi.org/10.1021/acs.analchem.5c04847
  17. Clin Chim Acta. 2025 Nov 26. pii: S0009-8981(25)00620-5. [Epub ahead of print] 120741
    Liquid chromatography-mass spectrometry quantitation and prevalence of medetomidine and xylazine in New Haven, Connecticut.
    Jillian Kodger, Gina Cassella-McLane, Mallorie Iozzo, Joe M El-Khoury.
      The emergence of veterinary sedatives such as xylazine and medetomidine as adulterants in the illicit drug supply poses increasing challenges to clinical toxicology and public health. Medetomidine, a potent alpha-2 adrenergic receptor agonist not approved for human use, has recently been detected in overdose cases, particularly in fentanyl-positive individuals. To address this, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay capable of detecting xylazine and medetomidine in urine with minimal sample preparation, a 60-min hydrolysis incubation, and a 7-min chromatographic run time. Analytical validation demonstrated acceptable precision, accuracy, linearity, and minimal matrix effects. We then analyzed 100 retrospective urine samples from a polydrug-using population. Xylazine was detected in 20 % and 3-hydroxymedetomidine (medetomidine's primary metabolite in urine) in 12 % of samples, with both compounds most frequently co-occurring with fentanyl. A subset of medetomidine-positive cases also tested positive for xylazine, indicating potential co-adulteration. This study highlights the value of expanded toxicological testing in identifying emerging adulterants that may otherwise go undetected by routine screening.
    Keywords:  LC-MS/MS and adulterant; Medetomidine; Xylazine
    DOI:  https://doi.org/10.1016/j.cca.2025.120741
  18. Anal Chem. 2025 Nov 27.
    RSR-MSI: Reference-Based Super-Resolution for Mass Spectrometry Imaging of Tissues and Single Cells.
    Yifan Fang, Cipeng Wu, Chentao Zhang, Yizhu Xu, Zhibin Yin, Zhouyi Xu, Wei Hang.
      High-spatial-resolution mass spectrometry imaging (MSI) visualizes molecular distributions in tissues and cells. However, achieving higher spatial resolution typically necessitates smaller pixel dimensions and an increased number of pixels, leading to longer data acquisition times and diminished analytical throughput. Although deep learning approaches have demonstrated significant potential in MSI, they typically require large training data sets or paired images, which are often unavailable. Herein, we propose the reference-based super-resolution for mass spectrometry imaging (RSR-MSI) method, with optical microscopy images as reference frames to extract abundant texture information. By integrating this with ion intensity data from the original MS images, we develop an image-specific super-resolution network. Employing solely a single low-resolution MS image coupled with a reference optical image, we successfully reconstruct high-resolution MS images for biological tissues and single cells, producing results with rich chemical and textural details. This approach significantly decreases the routine pixel-by-pixel scanning time by an order of magnitude while achieving high spatial resolution using existing mass spectrometry instruments without any customized modifications. Overall, our work introduces and validates the application of image super-resolution methods within the realm of single-cell MSI at subcellular resolution, paving the way for the development of high-spatial-resolution and high-throughput MSI for cellular biology research.
    DOI:  https://doi.org/10.1021/acs.analchem.5c05933
  19. Metabolites. 2025 Nov 18. pii: 749. [Epub ahead of print]15(11):
    LC-MS/MS Detection of Tryptophan, Kynurenine, Kynurenic Acid, and Quinolinic Acid in Urine Samples from Drug-Positive and Illicit Drug-Negative Patients with a Known History of Substance Use Disorder.
    Lindsey Contella, Christopher L Farrell, Luigi Boccuto, Alain H Litwin, Hunter Flanagan, Stacy E F Melanson, Nicole V Tolan, Marion L Snyder, Dina N Greene.
       INTRODUCTION: Currently, there are few tools for monitoring recovery in substance use disorder. As substance use has increased in prevalence, tools for measuring recovery are needed to improve therapeutic outcomes. Measuring the kynurenine pathway for imbalances in metabolites could be a possible solution to monitor recovery.
    METHODS: We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify tryptophan, kynurenine, kynurenic acid, and quinolinic acid in urine. Metabolites were separated using a stepwise gradient and detected with an Agilent 6460 triple quadrupole mass analyzer. The samples were extracted using a simple protein precipitation protocol. Method validation was performed using routine toxicology urine samples and laboratory contrived samples. The performance characteristics assessed included precision, linearity, stability, interference, and matrix effects. Additionally, urine samples from two cohorts (illicit drug-negative and drug-positive; n = 120 per cohort) were analyzed for significant concentration differences in the four metabolites using Mann-Whitney, PCA, and Area Under the Receiver Operating Characteristic Curve statistical analysis.
    RESULTS: The LC-MS/MS assay was linear from 195 to 100,000 ng/mL for tryptophan, 6 to 3000 ng/mL for kynurenine, 14 to 7200 ng/mL for kynurenic acid, and 125 to 64,000 ng/mL for quinolinic acid using an 8-point calibration curve. Imprecision ranged from 1.17% to 12.46% CV using two controls that spanned the analytical measurement range. Matrix effects were observed; however, the use of labeled internal standards matching the metabolites of interest minimized the impact on quantification. The extraction recovery efficiency was acceptable for the analytical validation. Ambient stability extended to 10 days, resulting in individual sample biases of up to 22%. A statistically significant increase in TRP, KYN, and QA was observed in drug-positive urine compared to illicit drug-negative urine (p < 0.01).
    CONCLUSION: We developed a rapid and sensitive LC-MS/MS method for quantifying tryptophan, kynurenine, kynurenic acid, and quinolinic acid in urine that can aid in future research elucidating the relationship between substance use disorders and tryptophan metabolism.
    Keywords:  LC-MS/MS; kynurenine; serotonin; substance use disorder; tryptophan; urine
    DOI:  https://doi.org/10.3390/metabo15110749
  20. Front Public Health. 2025 ;13 1687056
    AI redefines mass spectrometry chemicals identification: retention time prediction in metabolomics and for a Human Exposome Project.
    Fenna C M Sillé, Carsten Prasse, Thomas Luechtefeld, Thomas Hartung.
      The comprehensive identification of environmental and endogenous chemicals in human biospecimens is a critical bottleneck for realizing the Human Exposome Project. Untargeted metabolomics, particularly liquid chromatography-high-resolution mass spectrometry (LC-HRMS), offers unparalleled coverage of small molecules, but most detected features remain unidentified due to limited spectral libraries and structural ambiguity. Retention time (RT) prediction-based on quantitative structure-retention relationships (QSRR) and enhanced by artificial intelligence (AI)-is an underutilized orthogonal parameter that can substantially improve metabolite annotation confidence. This review synthesizes advances in machine learning-based RT prediction, probabilistic calibration, and cross-platform harmonization for liquid chromatography and gas chromatography, including deep learning, graph neural networks, and transfer learning approaches. We evaluate workflows integrating RT prediction with mass-based searches and network-based annotation tools, highlighting their potential to refine candidate ranking and reduce false positives in environmental exposure assessment. The use of endogenous compounds as internal calibrants is discussed as a practical strategy for improving RT transferability across laboratories. We further outline how RT-aware annotation supports non-targeted screening of emerging contaminants, transformation products, and exposure biomarkers, thereby enhancing the interpretability and reproducibility of exposomics data. By integrating RT prediction, QSRR modeling, and AI into untargeted metabolomics pipelines, researchers can move from qualitative detection toward quantitative, inference-driven mapping of environmental influences on human health, strengthening the scientific foundation for environmental health policy and preventive public health strategies.
    Keywords:  Human Exposome Project; artificial intelligence; environmental health; exposomics; quantitative structure–retention relationships; retention time prediction; untargeted metabolomics
    DOI:  https://doi.org/10.3389/fpubh.2025.1687056
  21. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Nov 21. pii: S1570-0232(25)00421-0. [Epub ahead of print]1269 124867
    Development and validation of an LC-MS/MS method for the simultaneous detection of urinary inflammatory biomarkers in a Flemish birth cohort.
    Fatima den Ouden, Adam Cseresznye, Liesa Engelen, Elias Maris, Ellen De Paepe, Giulia Poma, Sebastian Proost, Arnau Vich I Vila, Lieselot Y Hemeryck, Roger Pero-Gascon, Sarah De Saeger, Jeroen Raes, Lynn Vanhaecke, Tim S Nawrot, Adrian Covaci.
      Chronic inflammation is a significant contributor to various diseases but its assessment via blood sampling presents challenges, particularly in children. The evaluation of urinary biomarkers, including 3-bromotyrosine (Bty), 3-chlorotyrosine (Cty) and leukotriene E4 (LTE4), offers a non-invasive alternative. This study presents the optimization and validation of a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Bty, Cty and LTE4 in urine. Under optimized conditions, sample preparation was based on SPE using Oasis MAX cartridges, followed by LC-MS/MS analysis. Method performance was validated using the ICH 10 guidelines, resulting in satisfactory results for all analytes in terms of recovery, linearity, limits of quantification, precision and accuracy. Recovery rates ranged from 82 % to 97 %, while matrix effects were observed within the range of -11 % to 26 %. Linear range spanned from 0.08 to 20 ng/mL for the three analytes. Application to 332 urine samples from the ENVIRONAGE birth cohort (Belgium), comprising of children aged 4-11 years, revealed detection frequencies of 18 % for LTE4, 19 % for Cty and 50 % for Bty. Notably, creatinine-corrected Cty and LTE4 exhibited statistically significant Spearman correlations with established systemic inflammation markers. Specifically, Cty was positively correlated with absolute monocyte count (ρ = 0.53, p < 0.05), while LTE4 showed a positive correlation with relative eosinophil levels (ρ = 0.46, p < 0.05) and a negative correlation with the relative neutrophil levels (ρ = -0.56, p < 0.01). These results highlight the validated method as a valuable tool for investigating distinct inflammatory pathways in epidemiological settings and clinical research.
    Keywords:  3-bromotyrosine; 3-chlorotyrosine; Inflammation; Leukotriene E4; Pediatric population; Urine
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124867
  22. J Chromatogr Sci. 2025 Nov 15. pii: bmaf057. [Epub ahead of print]63(10):
    Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Quantification of 3,14,19-Triacetyl Andrographolide in Rat Plasma: Application to Preclinical Pharmacokinetic Studies.
    Chang Ren, Xin Sui, Kunlin Zhang, Jingjing Wang, Hao Li, Fenghe Yang, Mengjun Zhang, Junli Zhao, Yu Zhang, Guanhua Du, Guang Han.
      3,14,19-triacetyl andrographolide (ADA), a derivative of andrographolide, has emerged as a promising anti-inflammatory compound. In the present study, we established a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of ADA in rat plasma. ADA and celecoxib (internal standard, IS) were separated using an ACQUITY UPLC BEH C18 chromatographic columns with a gradient mobile phase of acetonitrile (containing 0.1% formic acid)/water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Quantification was performed using the multiple reaction monitoring transitions to m/z 499.1 → 439.1 for ADA, m/z 381.9 → 361.9 for celecoxib, with plasma samples undergoing liquid-liquid extraction. The method effectively detected ADA in plasma and exhibited excellent linearity (R 2 > 0.9921) over concentrations ranging from 5 to 2000 ng/mL. Both within-run and between-run precision (%RSD) were less than 6.88%, and accuracy ranged from 94.86% to 107.61%. Matrix effects were 87.53% and 112.78%, with recoveries ranging from 98.60% to 104.39%. ADA was proven to be stable under different storage conditions (4°C and -70°C) in plasma. The validated method was successfully applied to determine ADA concentrations in rat plasma. The results demonstrated that ADA exhibited a noticeable improvement in bioavailability compared with andrographolide.
    DOI:  https://doi.org/10.1093/chromsci/bmaf057
  23. Steroids. 2025 Nov 24. pii: S0039-128X(25)00165-5. [Epub ahead of print]225 109724
    LC-MS/MS quantitation of the primary reduced metabolites of progesterone in serum during the third trimester of human pregnancy reveals a potential role for 20β-hydroxyprogesterone and 5β-dihydroprogesterone in functional progesterone withdrawal.
    Edward Hinchliffe, Alexander Heazell.
      Progesterone (P4) is an essential steroid hormone synthesised by the placenta required for the maintenance of pregnancy. In humans, the metabolism of P4 has been implicated in functional P4 withdrawal prior to parturition. We have developed a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantitation in human serum of pregnenolone, P4 and its four primary reduced metabolites; 20α-hydroxyprogesterone (20α-OHP), 20β-hydroxyprogesterone (20β-OHP), 5α-dihydroprogesterone (5α-DHP) and 5β-dihydroprogesterone (5β-DHP). Following solid phase extraction, chromatographic baseline separation of each steroid was achieved using a biphenyl stationary phase within a 10.0 min runtime, followed by MS detection on a Sciex 6500+. The LC-MS/MS method was validated in accordance with published guidelines, confirming acceptable analytical performance pertaining to linearity, imprecision, accuracy, sensitivity, matrix effects, specificity and carryover. The method was applied to a large cohort of third trimester pregnancies with verified uncomplicated neonatal outcomes. Maternal circulating concentrations of P4, 20α-OHP, 20β-OHP, and 5α-DHP positively correlated with fetal gestational age. The ratio of P4:20β-OHP declined significantly throughout the third trimester, whilst the ratio of P4:5β-DHP increased at full term from 40 weeks' gestation. These findings may indicate a substantive role for β-reduction of P4 in the mechanics of functional P4 withdrawal, either via depletion of the overall pool of bioactive P4 or competitive binding of these metabolites to the P4 receptor in maternal and fetal tissue. Additionally, detailed characterisation of the normal maternal steroidome will facilitate the study of dysregulated placental steroidogenesis, which has been implicated in the pathogenesis of the major obstetric syndromes causing poor pregnancy outcomes.
    Keywords:  20β-Hydroxyprogesterone; 5α-Dihydroprogesterone; 5β-Dihydroprogesterone 20α-Hydroxyprogesterone; Liquid chromatography tandem mass spectrometry; Pregnenolone; Progesterone
    DOI:  https://doi.org/10.1016/j.steroids.2025.109724
  24. J Am Soc Mass Spectrom. 2025 Nov 25.
    High-Throughput Mass Spectral Library Searching of Small Molecules in R with NIST MSPepSearch.
    Andrey Samokhin, Mikhail Khrisanfov.
      High-level programming languages such as Python and R are widely used in mass spectrometry data processing, where library searching is a standard step. Despite the availability of numerous library search algorithms, those developed by NIST and implemented in MS Search remain predominant, partly because commercial databases (e.g., NIST, Wiley) are distributed in proprietary formats inaccessible to custom code. MSPepSearch, another NIST tool, provides access to the same algorithms with greater flexibility for automation. However, its use requires calling a command-line interface with multiple flags and parsing output text files to retrieve results, which can be cumbersome. To address this, we developed mspepsearchr, an R package that streamlines the integration of library searches against NIST-format mass spectral databases into complex, multistep workflows. MSPepSearch is a single-threaded tool; therefore, parallelization was achieved externally by running multiple instances from within R. We describe the package, evaluate its performance, and illustrate its utility through the recognition of steroid-like compounds in untargeted gas chromatography-mass spectrometry analysis of biological samples.
    DOI:  https://doi.org/10.1021/jasms.5c00322
  25. J Chromatogr A. 2025 Nov 17. pii: S0021-9673(25)00895-7. [Epub ahead of print]1765 466551
    A widely targeted analytical method for oxylipins including enantiomers using chiral liquid chromatography tandem mass spectrometry.
    Yutaka Umakoshi, Yoshihiro Izumi, Tomomi Hashidate-Yoshida, Hideo Shindou, Takeshi Bamba.
      Oxylipins are lipids formed when polyunsaturated fatty acids are oxidized. Several oxylipins play important roles in the body, such as regulation of inflammatory responses. Although oxylipin measurement is commonly performed using reversed-phase liquid chromatography tandem mass spectrometry, conventional methods cannot resolve enantiomers. The aim of this study was to develop a widely targeted analysis method for oxylipins that could separate enantiomers. First, seven polysaccharide-derived chiral columns were screened, and the CHIRALPAK IG column, which achieved the highest resolution of eicosanoid isomers, was selected. Next, the set of targeted compounds was expanded, and the chromatographic separation of 151 compounds, including 40 pairs of enantiomers, was evaluated. The constructed method successfully separated 55 out of 59 pairs of isomers, including enantiomers. Finally, the established method was used to analyze mouse plasma and five types of tissues, and approximately 100 compounds exhibiting characteristic profiles were detected in each tissue. After evaluating hydroxyeicosatetraenoic acids (HETEs), monohydroxy compounds of arachidonic acid, it was found that the S-enantiomer of 12-HETE was more prevalent than the R-enantiomer in the lungs and spleens. Conversely, R-enantiomer of 9-HETE and 11-HETE was more abundant than the S-enantiomer. The amounts of R- and S-enantiomers of 5-HETE and 8-HETE were nearly equal in certain tissues. This analytical method is useful for biomarker discovery and accurately understanding the pathways of oxylipin formation because it can perform a widely targeted analysis of oxylipins, including enantiomers.
    Keywords:  Chiral separation; LC/MS; Lipidomics; Oxylipin; Widely targeted analysis
    DOI:  https://doi.org/10.1016/j.chroma.2025.466551
  26. Methods Mol Biol. 2026 ;2994 131-144
    Analysis of the Isomers of New Chiral Psychoactive Substances Using Enantioselective High-Performance Liquid Chromatography-Mass Spectrometry.
    Giorgia Sprega, Giorgi Kobidze, Alfredo Fabrizio Lo Faro, Francesco Paolo Busardo, Bezhan Chankvetadze.
      The rapid emergence of new chiral psychoactive substances makes the simultaneous separation of enantiomers and structural isomers crucial for distinguishing compounds with identical molecular masses but with different pharmacological and toxicological properties, as well as frequently with a different legal status. Enantioselective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) plays a key role in identifying new chiral psychoactive substances, profiling their distinct pharmacokinetics, and supporting regulatory and forensic efforts. Despite its high sensitivity and accuracy, a mass spectrometer cannot differentiate enantiomers, and a differentiation of positional isomers is often challenging. Therefore, enantiomers and/or positional isomers have to be pre-separated prior to entering the mass spectrometer. This chapter describes the simultaneous separation and enantioseparation of positional isomers of chloromethcathinones (CMCs) and methylmethcathinones (MMCs).
    Keywords:  Chirality; Enantioseparation; HPLC–MS/MS; New chiral psychoactive substances; Separation of stereo- and structural isomers
    DOI:  https://doi.org/10.1007/978-1-0716-5023-3_7
  27. bioRxiv. 2025 Nov 13. pii: 2025.11.12.688047. [Epub ahead of print]
    Learning from All Views: A Multiview Contrastive Framework for Metabolite Annotation.
    Yan Zhou Chen, Soha Hassoun.
      Metabolomics, enabled by high-throughput mass spectrometry, promises to advance our understanding of cellular biochemistry and guide new discoveries in disease mechanisms, drug development, and personalized medicine. However, as the assignment of molecular structures to measured spectra is challenging, annotation rates remain low and hinder potential advancements. We present MultiView Projection (MVP), a novel framework for learning a joint embedding space between molecules and spectra by leveraging multiple data views: molecular graphs, molecular fingerprints, spectra, and consensus spectra. MVP builds on contrastive multiview learning to capture mutual information across views, leading to more robust and generalizable representations for spectral annotation. Unlike prior approaches that consider multiple views via concatenation or as targets of auxiliary tasks, MVP learns from all views jointly, resulting in improved molecular candidate ranking. Notably, MVP supports annotation using either individual spectra or consensus spectra, enabling flexible use of multiple measurements. On the MassSpecGym benchmark, we show that annotation using query consensus spectra significantly outperforms rank aggregation strategies based on constituent spectrum annotation. Using the consensus spectrum view, MVP achieves 35.99% and 13.96% rank@1 when retrieving candidates by mass and formula, respectively. When ranking using individual spectra, MVP demonstrates performance that is superior to or on par with existing methods, achieving 26.37% and 11.10% rank@1 for candidates by mass and formula, respectively. MVP offers a flexible, extensible foundation for learning from multiple molecule/spectra data views.
    For Table of Contents Only:
    DOI:  https://doi.org/10.1101/2025.11.12.688047
  28. Anal Chim Acta. 2026 Jan 01. pii: S0003-2670(25)01199-7. [Epub ahead of print]1381 344805
    Liver microsomal fraction induced small molecule isotope labeling via catalyzed H/D and 16O/18O isotope exchange reactions: A novel approach for obtaining isotope-labeled compounds.
    Boris Tupertsev, Sergey Osipenko, Tatiana Ikonnikova, Yury Kostyukevich.
       BACKGROUND: The use of stable isotope-labeled compounds in various omics studies, combined with chromatography-mass spectrometry (MS) techniques, has become a powerful strategy to enhance quantitative accuracy, identification confidence, and biological insight. However, current labeling strategies are often limited by high cst, operational complexity, and narrow applicability. Consequently, there is a growing need for novel methods that address both qualitative and quantitative challenges in biological sample analysis. As a promising alternative, this study explores an enzymatic labeling approach using liver microsomes as biocatalysts for stable isotope incorporation into small molecules.
    RESULTS: We developed an enzymatic isotope labeling method using rat liver microsomal fraction to label progesterone and seven structurally related compounds with deuterium and oxygen-18. Labeling was performed in 50 mM phosphate-buffered saline (PBS, pH 7.4) using 99.8 % D2O and 50 % H218O at 37 °C in the absence of MgCl2 and NADPH. Compared to non-enzymatic controls, hydrogen/deuterium and oxygen-16/oxygen-18 exchanges were accelerated by up to 32- and 41-fold, respectively. Structure-dependent labeling patterns and mechanisms, as well as the rates and extents of heavy isotope incorporation, were evaluated using HPLC-MS/MS and NMR. Notably, 0.3 mg of deuterium-labeled progesterone was obtained in under two weeks without the need for total synthesis, demonstrating the method's practical utility and scalability for standard production.
    SIGNIFICANCE: This low-cost enzyme-based labeling method facilitates rapid identification of functional groups and efficient preparation of labeled standards for quantitative MS analyses. It offers a practical, scalable alternative to traditional synthetic labeling techniques. With minimal setup and broad compounds applicability, the approach provides significant advantages for MS-based omics workflows in both research and applied analytical laboratories.
    Keywords:  Internal standards; Liver microsomes; Mass spectrometry; Omics studies; Stable isotope labeling
    DOI:  https://doi.org/10.1016/j.aca.2025.344805
  29. Molecules. 2025 Nov 14. pii: 4404. [Epub ahead of print]30(22):
    Simultaneous Quantification of Multiple Analytes in Rat Plasma by UHPLC-MS/MS Following Oral Administration of Gastrodiae Rhizoma Extract for Pharmacokinetic Evaluation.
    Lu Chen, Yameng Zhu, Huizi Ouyang, Xiwei Wu, Wenhan Lin, Kaili Zhang, Jun He.
      Gastrodiae Rhizoma (GR) is known to have a medicinal and food-based homology. It is used to treat infantile convulsion, epilepsy, spasm, tetanus, and vertigo. In this study, an ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated to quantify fourteen components (p-hydroxybenzyl alcohol, gastrodin, parishin E, p-hydroxybenzoic acid, parishin C, parishin A, parishin B, nicotinamide, p-hydroxybenzaldehyde, adenosine, 3,4-dihydroxybenzaldehyde, syringaldehyde, dauricine, and nobiletin) of GR in rat plasma. Methanol precipitation was used to prepare the samples with astragalin, serving as the internal standard. In multiple reaction monitoring (MRM) mode, the fourteen components were separated by gradient elution on a Waters ACQUITY UPLC® HSS T3 column. Under these conditions, all fourteen analytes' calibration curves demonstrated strong linearity within wide concentration ranges (r > 0.9941). Accuracy for the intra-day and inter-day assessments ranged from -13.74% to 12.76%, and the precision for all analytes remained below 8.88%. The analytes' extraction recoveries ranged from 66.78% to 114.2%, accompanied by matrix effects ranging from 63.65% to 117.61%. Under the evaluated conditions, stability tests confirmed that the compounds remained stable, with relative standard deviations below 13.83%. Consequently, the UHPLC-MS/MS method was effectively used to determine the pharmacokinetics of fourteen components in rat plasma after oral administration of GR extract. This study provides supportive data for rational application of GR.
    Keywords:  Gastrodiae Rhizoma extract; UHPLC-MS/MS; medicine food homology; pharmacokinetics; rat plasma
    DOI:  https://doi.org/10.3390/molecules30224404
  30. J Proteome Res. 2025 Nov 24.
    Storing Mass-Spectrometry Data in Simple Databases Enables Flexible and Intuitive Exploration without Time or Space Penalties.
    William Kumler, Sam LaRue, Anitra E Ingalls.
      Mass spectrometry (MS) generates large data sets that are stored in increasingly optimized and complex file types, demanding technical expertise to extract information rapidly and easily. We wondered whether a simple structured query language (SQL) database could hold raw MS data and allow for easily readable queries without incurring major penalties in the read time or disk space relative to other popular MS formats. Here, we describe a basic MS schema with intuitive database tables and fields that can outperform other formats for exploratory and interactive analysis according to six data subsets commonly extracted: single scans (both MS1 and MS2), ion chromatograms, retention time ranges, and fragmentation searches (both precursor and fragment search). Additionally, we compare SQLite, DuckDB, and Parquet implementations and find that they can perform these tasks in under a second, even when the files occupy over a gigabyte of data on the disk. We believe that this tidy data schema expands nicely to most forms of MS data and offers a way to transparently query data sets while preserving computational performance.
    Keywords:  SQL; benchmarking; data storage; exploratory data analysis; human-centered design; liquid chromatography; mass spectrometry
    DOI:  https://doi.org/10.1021/acs.jproteome.5c00721
  31. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Nov 21. pii: S1570-0232(25)00420-9. [Epub ahead of print]1269 124866
    Quantification of a sea lamprey (Petromyzon marinus) pheromone antagonist in river water using ion pairing solid phase extraction coupled with liquid chromatography-tandem mass spectrometry.
    Anne M Scott, Sonam Tamrakar, Weiming Li.
      Pheromones mediate species-wide communication for many aquatic organisms, and the measurement of pheromones in natural waters is essential to understanding the environmental context of their function. However, chemical measurement of environmental pheromones and their antagonists is technically demanding and remains underdeveloped relative to assays for characterizing biological functions and application efficacy. In this study, we developed and validated an accurate and sensitive method to quantify a sea lamprey (Petromyzon marinus) pheromone and its antagonists. In this species, males release a multi-component sex pheromone containing 3-keto petromyzonol sulfate (3kPZS) that attracts females, while related compounds petromyzonol sulfate (PZS) and petromyzonol tetrasulfate (3sPZS) antagonize and disrupt female attraction. Developing methods to quantify 3sPZS in river water that contains pheromone is essential for understanding concentration-dependent effects of antagonists on invasive sea lamprey spawning. The target compound 3sPZS was extracted using triethylamine as an ion-pairing reagent during solid phase extraction followed by quantification using liquid chromatography-tandem mass spectrometry. The method showed a limit of detection of 0.1 ng/mL and limit of quantification of 0.5 ng/mL with linearity in the range of 10-1000 ng/mL. The intra- and inter-day accuracy, precision, recovery, and matrix effect of this method were evaluated. The method was applied to quantify 3sPZS, PZS, and 3kPZS in water sampled during field application in a river with sea lamprey and further evaluated for robustness by quantifying 3sPZS in 16 rivers across diverse environmental matrices. Our approach may be adapted to inform management strategies for detecting and mitigating invasive or imperiled aquatic species.
    Keywords:  Bile acid; Invasive species; LC-MS/MS; Petromyzonol tetrasulfate; Sea lamprey; Solid phase extraction; Triethylamine
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124866
  32. J Chromatogr A. 2025 Nov 17. pii: S0021-9673(25)00894-5. [Epub ahead of print]1766 466550
    Current trends in liquid chromatography-mass spectrometry analysis of small interfering RNAs: A short review.
    Hiroyuki Togawa, Takao Yamaguchi, Junji Kawakami, Satoshi Obika.
      Small interfering RNA (siRNA) therapeutics represent one of the most rapidly advancing pharmaceutical modalities. Since the approval of the first siRNA-based drug in 2018, seven siRNA therapeutics have been approved to date. During manufacturing, "non-optimal duplexes" and "single-stranded impurities" can arise from synthetic byproducts such as shortmers and longmers, or from minor imbalances in strand quantification and mixing. Furthermore, phosphorothioate linkages introduce chirality, generating complex diastereomeric mixtures. The use of delivery systems, including lipid nanoparticles or N-acetylgalactosamine (GalNAc) conjugates, further increases the structural complexity and heterogeneity of the final product. Compared to single-stranded oligonucleotides, siRNA-being double-stranded-yields more complex chromatographic and mass spectral profiles, making analytical characterization particularly challenging. Therefore, the development of robust and reliable analytical methods is essential to ensure the quality of siRNA therapeutics. This review summarizes key analytical techniques for the quality evaluation of siRNA therapeutics. Liquid chromatography (LC) is one of the most widely used approaches, with separation modes such as ion-pair reversed-phase (IP-RP), anion exchange (AEX), size exclusion chromatography (SEC), and hydrophilic interaction chromatography (HILIC) being actively explored. Mass spectrometry (MS) is another powerful tool that provides molecular mass-based structural information not accessible via UV detection alone. The combination of LC and MS offers a highly effective platform for characterizing siRNA therapeutics, enabling both qualitative and quantitative assessment of impurities and isomers. Furthermore, tandem MS (MS/MS) can provide sequence-specific information for the identification of active pharmaceutical ingredients. In addition to quality assessment, the application of LC and MS techniques in the pharmacokinetic analysis of siRNA therapeutics is also briefly discussed.
    Keywords:  Liquid chromatography; Mass spectrometry; siRNA
    DOI:  https://doi.org/10.1016/j.chroma.2025.466550
  33. J Proteome Res. 2025 Nov 25.
    Targeted Metabolomic Strategy for Epigenetic Modification-Related Metabolites Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.
    Wenbin Cai, Weiyan Sun, Ziru Wu, Yang Bian, Zhongze Zhang, Qian Cheng, Xuelian Shi, Le Liu, Yi Zhu, Chunxiao Wan, Xiaohong Wang, Hongfeng Jiang, Xu Zhang.
      Epigenetics-related metabolites-substrates or cofactors that regulate epigenetic modifications, that play a critical role in regulating gene expression-are collectively referred to as the "epigenetic metabolome". Here, we developed a comprehensive targeted metabolomic method covering 33 metabolites involved in multiple types of epigenetic modifications. The detection panel included coenzyme A (CoA)/acetyl-CoAs-metabolites in the methionine cycle, those related to nicotinamide adenine dinucleotide (NAD+) metabolism─intermediates of carbohydrate metabolism, and acetylglucosamines. These metabolites were analyzed in two liquid chromatography-mass spectrometry runs based on their distinct chemical properties. For most metabolites (over 88%), the limits of quantification were below 16 ng, the dynamic ranges exceeded 3 orders of magnitude, and the precisions were above 80%. We profiled the epigenetic metabolome in a mouse model of diabetic cardiomyopathy and identified 8 significantly altered metabolites linked to various epigenetic modifications, including DNA/histone methylation, acetylation, and O-GlcNAcylation of histones. In conclusion, we established a reliable and sensitive method for detecting alterations in the epigenetic metabolome and demonstrated its applicability to disease-related studies.
    Keywords:  coenzyme; diabetic cardiomyopathy; epigenetics; metabolomics
    DOI:  https://doi.org/10.1021/acs.jproteome.5c00296
  34. Forensic Sci Med Pathol. 2025 Nov 25.
    Dual one-step LC-MS/MS methods for hair analysis in drug-facilitated crime: application to an alleged Fraud-Related case.
    Ismail Ethem Goren, Nebile Daglioglu.
      This study aimed to develop and validate two simplified, one-step extraction methods coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous segmental analysis of psychoactive substances, specifically doxylamine (DOX), haloperidol (HAL), citalopram (CTP), sildenafil (SDF), and common illicit drugs in hair samples. A secondary objective was to apply these methods to a real-life forensic case involving suspected prolonged drug-facilitated crime (DFC) with suspected non-consensual exposure with financial implications.Two analytical methods based on one-step extraction protocols coupled with LC-MS/MS were developed and validated according to ANSI/ASB 036 standards. One method was based on ultrasonic solvent extraction (USE), while the other relied on passive solvent incubation (PSI). Hair samples from two victims were collected and segmented to assess chronic drug exposure. Analytical performance was evaluated in terms of linearity, sensitivity, accuracy, precision, matrix effects, recovery, and dilution integrity.Both methods demonstrated high sensitivity (LODs as low as 0.27 pg/mg), accuracy (bias within ± 15%), and precision (RSD ≤ 18.3%). Segmental analysis of Victim A's hair revealed DOX and HAL concentrations consistent with chronic, non-consensual administration. HAL was also detected in Victim B's scalp and leg hair, while DOX was absent. The segmental distribution patterns supported the hypothesis of prolonged sedative non-consensual drug exposure.This study presented a rare case of drug-facilitated crime involving chronic administration of HAL and DOX within an alleged deception-based context. The validated LC-MS/MS methods proved to be robust, cost-effective, and suitable for routine forensic toxicology. Segmental hair analysis provided critical retrospective evidence, reinforcing its value in complex DFC investigations. The interpretation remained confined to analytical evidence, without inferring intent.
    Keywords:  Antipsychotics; Doxylamine; Drug facilitated crimes; Forensic; Fraud; Hair; Haloperidol
    DOI:  https://doi.org/10.1007/s12024-025-01137-x
  35. Mass Spectrom Rev. 2025 Nov 27.
    Mass Spectrometry-Based Applications in Tannin Analytics: From Qualitative and Quantitative Analyses to Biological Activity.
    Marica T Engström, Maarit Karonen.
      Tannins are widespread specialized plant metabolites that contribute significantly to the polyphenol content of plant-based diets. Their effects on human and animal health vary depending on their structure, with potential benefits including antioxidative, antimicrobial, anthelmintic, and anticarcinogenic properties. Understanding tannin composition and quantity in plant products is essential, as their bioactivities are influenced by their functional groups. Mass spectrometry-based techniques excel in tannin analysis, offering both qualitative and quantitative insights. Combining ultrahigh-performance liquid chromatography with electrospray ionization and high-resolution and triple quadrupole mass analyzers is optimal for comprehensive tannin profiling. Such an approach enables precise analysis and helps predict tannin bioactivities. This review highlights the mass spectrometric analysis of proanthocyanidins and hydrolysable tannins, addressing ionization techniques, interpretation of multiply charged ions, characteristic fragmentations, and reaction monitoring. Applications related to tannin bioactivities are also briefly discussed, demonstrating the utility of mass spectrometry in tannin analysis in complex sample matrices.
    Keywords:  bioactivity; electrospray ionization; fragmentation patterns; high‐resolution mass spectrometry; multiple reaction monitoring
    DOI:  https://doi.org/10.1002/mas.70013
  36. Molecules. 2025 Nov 20. pii: 4478. [Epub ahead of print]30(22):
    A Novel and Validated GC-MS/MS Method for the Detection of Four Opioids and Seven Fentanoids in Oral Fluid for Forensic Applications.
    Roberta Tittarelli, Davide Filardi, Federico Mineo, Giulio Mannocchi.
      In recent years, the marked increase in the abuse of fentanyl and its analogues has emphasized the importance of developing highly sensitive and selective analytical methods for their detection in biological matrices. Oral fluid (OF) has emerged as a useful alternative to blood in forensic toxicology, offering a non-invasive and easily accessible matrix for the identification of a recent drug intake. However, its composition requires rigorous sample preparation and robust analytical techniques. A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed and validated for the quantification of four opioids and seven fentanyl analogues. A fast and simple solid-phase extraction (SPE) procedure was optimized, enabling the identification and quantification of all analytes in 11 min. The method was validated according to international guidelines, showing a satisfactory degree of linearity (R2 ≥ 0.993), precision, accuracy, and sensitivity, with limit of detections (LODs) ranging from 0.10 to 0.20 ng/mL. The method was then successfully applied to n = 10 real OF samples collected during traffic stops set up by police forces which tested negative at the screening tests. Two samples tested positive for codeine and morphine, and one was positive for fentanyl and norfentanyl. The small number of samples currently limits the interpretation of the results. However, our study represents a good starting point for further application of this method to a wider population of real samples.
    Keywords:  fentanyl; gas chromatography; mass spectrometry; opioids; oral fluid; toxicology
    DOI:  https://doi.org/10.3390/molecules30224478
  37. Commun Biol. 2025 Nov 25. 8(1): 1673
    Metabolomic and lipidomic atlas of human hair across its length.
    Maria van de Lavoir, Yuanye Chi, Ting Zeng, Rani Robeyns, Maria Del Mar Delgado Povedano, Leen Jacobs, Celine Gys, Hugo Neels, Shuzhao Li, Alexander L N van Nuijs, Adrian Covaci.
      The stable structure and growth cycle of human hair enables the accumulation of both endogenous and exogenous compounds, making hair an ideal matrix for long-term profiling. However, its exact molecular composition, shaped by root-to-tip variations, remains poorly understood. Here, we present a metabolome and lipidome atlas of human hair originating from a healthy cohort. The Human Hair Atlas maps over 1200 unique molecular species and highlights compounds prone to significant longitudinal variations. Metabolite and lipid levels vary by up to 50% along the length of the hair, underscoring the importance of accounting for segmental differences. We categorized 122 molecules as exposome-related compounds, primarily from personal care products, facilitating biological interpretation. The Human Hair Atlas is publicly accessible ( https://metabolomics.cloud/hair/ ), providing a resource to stimulate research in hair metabolomics, lipidomics, and exposomics, and to explore the potential of hair as a complementary matrix in clinical studies.
    DOI:  https://doi.org/10.1038/s42003-025-09069-6
  38. Metabolites. 2025 Nov 18. pii: 750. [Epub ahead of print]15(11):
    Metabolomics and Pharmacometabolomics: Advancing Precision Medicine in Drug Discovery and Development.
    Eleni V Stolaki, Konstantina Psatha, Michalis Aivaliotis.
      Metabolomics and pharmacometabolomics are at the forefront of precision medicine, serving as powerful tools in drug discovery and development. These approaches help address critical challenges in the field, including high clinical trial failure rates, adverse drug reactions, and interindividual variability in drug response. Comprehensive metabolome profiling enables the elucidation of disease mechanisms, identification of drug targets, optimization of therapeutic strategies, and assessment of drug safety and efficacy. It also supports more informed clinical trial design. This review highlights the pivotal role of metabolomics in advancing precision medicine and aims to broaden the perspectives of emerging scientists entering this complex field. Key analytical techniques-namely mass spectrometry and nuclear magnetic resonance spectroscopy-are discussed for their respective strengths and limitations in metabolite identification, quantitation, and structural elucidation. Additionally, analytical separation technologies such as liquid and gas chromatography, ion mobility spectrometry, capillary electrophoresis, and supercritical fluid chromatography are explored for their potential to enhance metabolome coverage, improve analytical efficiency, and reduce costs. Ongoing advancements in instrumentation and computational tools are helping to overcome major challenges in metabolomics, including metabolome complexity, data analysis and integration, and biomarker validation. These developments continue to expand the applications of metabolomics and pharmacometabolomics in both preclinical and clinical research. Ultimately, this review underscores their translational potential in facilitating drug discovery, mitigating risks in clinical trials, and shaping the future of precision medicine.
    Keywords:  NMR; drug development; drug discovery; mass spectrometry; metabolomics; pharmacometabolomics; precision medicine
    DOI:  https://doi.org/10.3390/metabo15110750
  39. Molecules. 2025 Nov 19. pii: 4471. [Epub ahead of print]30(22):
    Green and Emerging Microextraction Strategies for Bioanalytical Determination of Hormones: Trends, Challenges, and Applications.
    David Vicente-Zurdo, Sonia Morante-Zarcero, Isabel Sierra.
      Accurate and sensitive determination of hormones in biological matrices is essential for clinical diagnostics, therapeutic monitoring, and endocrine research. However, hormone determination presents significant challenges due to their typically low concentrations, complex sample matrices, and structural diversity. In recent years, microextraction techniques have emerged as strategic tools in bioanalytical chemistry, offering advantages in terms of miniaturization, enhanced selectivity, and compatibility with the principles of green analytical chemistry (GAC). This review provides a comprehensive overview of green and emerging microextraction approaches for the determination of steroidal, thyroid, peptide, and other hormones in biological samples. Key techniques such as solid-phase microextraction (SPME) and dispersive liquid-liquid microextraction (DLLME), followed by high-performance liquid chromatography (HPLC) coupled to diode array detectors (DADs) or mass spectrometry (MS), are critically discussed. Special emphasis is placed on the use of environmentally friendly solvents, such as deep eutectic solvents (DESs), supramolecular solvents (SUPRASs), and advanced sorbents including molecularly imprinted polymers (MIPs) and nanostructured magnetic phases. Applications across various bioanalytical matrices (urine, plasma, serum, saliva, tissues…) are examined in terms of sensitivity, selectivity, and validation parameters. Finally, current challenges, method development gaps, and future directions are highlighted to support the continued advancement of sustainable hormone determination in complex biological systems.
    Keywords:  bioanalytical matrices; green analytical chemistry; green solvents; liquid chromatography; mass spectrometry; microextraction techniques; smart materials; steroidal hormones; thyroid hormones
    DOI:  https://doi.org/10.3390/molecules30224471
  40. BMC Pharmacol Toxicol. 2025 Nov 25. 26(1): 200
    Simultaneous determination of iloperidone and its metabolites in rat plasma using a novel UPLC-MS/MS method: an application for drug-drug interaction.
    Xiaohai Chen, Dongxin Chen, Hualu Wu, Hailun Xia, Tian Lan, Ren-Ai Xu.
      This study aimed to develop and validate a novel ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of iloperidone (ILP) and its metabolites P88, P95 in rat plasma and to investigate drug-drug interaction (DDI) between shikonin and ILP in Sprague-Dawley rats. The separation of the analytes was performed on a UPLC BEH C18 column in the mobile phase (acetonitrile and water with 0.1% formic acid) with the flow rate of 0.4 mL/min. The quantitative analysis was performed in positive ion mode. A total of 10 Sprague-Dawley rats were divided into two groups: control group (1.0 mg/kg ILP alone) and experimental group (20 mg/kg shikonin plus 1.0 mg/kg ILP) to investigate the influence of shikonin on ILP metabolism in rats. We successfully established a quick UPLC-MS/MS analytical method for simultaneously detecting ILP and its two metabolites in rat plasma. Linearity, matrix effect, recovery, accuracy, precision and stability of this quantitative method was satisfied with Food and Drug Administration (FDA) guidelines. In addition, in vitro studies demonstrated that shikonin significantly inhibited CYP3A4- and CYP2D6-mediated metabolism in both rat liver microsomes (RLM) and human liver microsomes (HLM). Furthermore, we found the main pharmacokinetic parameters of ILP, such as AUC(0-t) and the peak plasma concentration (Cmax), were obviously changed, which were about twice higher in experimental group than the values in control group. The data demonstrated that shikonin obviously changed the main pharmacokinetics of ILP and its metabolites in rats. In the future clinical use, we should pay more attention to the concomitant application of shikonin and ILP in humans.
    Keywords:  Iloperidone; Interaction; Pharmacokinetics; Rat; Shikonin
    DOI:  https://doi.org/10.1186/s40360-025-01037-4
  41. Mikrochim Acta. 2025 Nov 23. 192(12): 841
    Tunable dual-ligand ZIF-8-90 effervescent tablet-assisted dispersive solid phase extraction for monitoring of both PAHs and their hydroxyl metabolites: from external to internal exposure analysis.
    Yue-Dan Zhao, Ke-Yan Chen, Lei Xia, Chen-Yu Jin, Jun-Jian Zhao, Hai-Jie Li, Hou-Jun Xu, Man-Man Wang.
      Mixed-ligand metal organic frameworks have received great attentions as novel sorbents for sample preparation due to their fascinating structures and intriguing functionalities. Here, we report the fabrication of mixed-linker ZIF-8-90 s with tunable properties for effervescent tablet-assisted dispersive solid-phase extraction (ET-DSPE) of polycyclic aromatic hydrocarbons (PAHs) in environmental water before HPLC separation and hydroxyl metabolites of PAHs (OH-PAHs) in urine samples prior to LC-MS/MS. ZIF-8-90 s based effervescent tablets possessed the high affinity towards analytes via Zn-O bond and π-π interaction, and afforded a great excluding ability to interfering components from urine samples. ET-DSPE of PAHs from 15 mL water sample with ZIF-825-9075 gave enhancement factors of 122-155, linear ranges of 4-105 ng/L, detection limits (S/N = 3) of 1-8 ng/L, and recoveries of 81.1%-103%. For the determination of OH-PAHs, the proposed ET-DSPE method using ZIF-885-9015 allowed direct loading of 4 mL of crude human urine sample and provided the linearity range of 0.004-100 ng/mL, detection limits of 0.001-0.008 ng/mL, and recoveries of 82.1%-104%. In conclusion, ZIF-8-90-based ET-DSPE coupled with HPLC and LC-MS/MS were successfully applied to monitor both PAHs in environmental water and OH-PAHs in urine in a facile, efficient and reliable way.
    Keywords:  Effervescent tablet-assisted dispersive solid-phase extraction; Environmental water; HPLC; Hybrid zeolitic-imidazolate frameworks; Hydroxylated polycyclic aromatic hydrocarbons; LC-MS/MS; Polycyclic aromatic hydrocarbons; Urine analysis
    DOI:  https://doi.org/10.1007/s00604-025-07703-y
  42. Anal Chem. 2025 Nov 24.
    Multidimensional Mass Spectral Similarity Algorithm: Discriminate Disaccharide and Flavonoid Isomers Coupled with Online Energy-Resolved Acquisition and Electron Activation Dissociation.
    Fangzhou Geng, Bowen Zhou, Pengfei Yang, Li Zhang, Jing Zhang, Duobin Mao, Yinlong Guo.
      Structural isomers are critical analytes in the biological and chemical arenas. Despite the ability of tandem mass spectrometry to provide fragment ion information, their high structural similarity impedes confident identification. To address this, we developed a novel method leveraging energy-resolved mass spectrometry (ER-MS) of fragment ions generated by electron activation dissociation (EAD). EAD initiated rapid radical chain dissociation via electron excitation and removal mechanisms, delivering superior isomer discrimination compared to conventional collision-induced dissociation (CID). Subsequent energy-resolved analysis further enhanced the distinction by integrating these dissociation mechanisms. Our strategy employed a cosine-based multidimensional spectral similarity algorithm to visualize and quantify subtle spectral differences across multiple energies. This method successfully distinguished many types of isomers, such as linkage, composition, and conformation isomers in disaccharides and flavonoid glycosides and achieved 93.8% top-1 identification accuracy against an in-house library. When applied to pomelo peel and commercial beverages for key metabolite characterization, it provided 44.4-50.0% top-1 annotation accuracy across all detected interest features. These results demonstrate that the multidimensional similarity algorithm that combines EAD and ER-MS significantly advances the depth and accuracy of compound annotation.
    DOI:  https://doi.org/10.1021/acs.analchem.5c05440
  43. Toxins (Basel). 2025 Oct 30. pii: 538. [Epub ahead of print]17(11):
    Optimisation of a One-Step Reusable Immuno-Affinity Purification Method for the Analysis and Detection of Fumonisin Mycotoxins in Foods and Feeds.
    Christian Kosisochukwu Anumudu.
      Fumonisins are among the most prevalent mycotoxins in maize and maize-based products, posing significant food safety and public health risks due to their hepatotoxic, nephrotoxic, and potential carcinogenic effects. Given the strict regulatory limits set by the European Commission and Codex Alimentarius, the development of reliable, sensitive, and matrix-robust analytical methods remain a priority for routine monitoring in both food and feed systems. In this study, a reusable immuno-affinity purification methodology for the quantitative determination of fumonisin mycotoxins (FB1, FB2 and FB3) in foods and feeds (maize matrix) was developed. A single extraction protocol using 2% formic acid in water was employed, followed by cleanup with an immuno-affinity purification column and toxin elution by methanol/PBS (1:1, v/v). Detection and quantification of the mycotoxins was achieved by a normal phase ultra-high performance liquid chromatography coupled with electrospray ionisation triple quadrupole mass spectrometry (UHPLC/ESI-MS/MS). The chromatographic mobile phase utilised was a linear gradient of methanol/water containing 0.1% formic acid. The developed method has a limit of detection of 2.5 ng/g and a limit of quantification of 5 ng/g, all well below the European commission's guidance values of 1000 ng/g for corn destined for human consumption and 800 ng/g for maize-based breakfast cereals and snacks. While the recovery rates of the method in this study ranged from 65-70% for the three fumonisin analogues in solutions, when tested in maize matrix, recoveries were markedly lower (~30%) due to pronounced matrix suppression. Good repeatability (standard deviation <10%) was achieved for all the fumonisin analogues. The developed method, although quick and effective in solvent systems, suffered limitations to its practical usage due to matrix suppression of the extracts derived from the immuno-affinity purification column, thus significantly reducing the application of the method in measuring fumonisin mycotoxins in food and feed samples. Overall, the method was effective in quantification of fumonisin mycotoxins in solvent solutions but not in food and feed matrices, thus necessitating further optimisation for practical usage. The performance of the developed method was compared to a commercial lateral flow immunochromatographic assay which proved to be better than the developed method in the quantification of toxins in food matrices, as the commercial lateral flow immunochromatographic assay outperformed the developed method in maize matrices. These findings highlight the need for matrix-based validation and further refinement of antibody stability to ensure robust application in regulatory monitoring of fumonisins using immunoaffinity purification methods.
    Keywords:  Fumonisin; Immuno-affinity purification; Maize; Mycotoxicosis; Tandem mass spectrometry; Ultra-high performance liquid chromatography
    DOI:  https://doi.org/10.3390/toxins17110538
  44. Talanta. 2025 Nov 22. pii: S0039-9140(25)01641-8. [Epub ahead of print]299 129150
    Lipidome mapping of natural rubber: Detailed investigation into the influence of geographical origin and treatment using liquid chromatography and high-resolution tandem mass spectrometry.
    Ludovica Sofia Guadalupi, Cosima Damiana Calvano, Tommaso R I Cataldi, Andrea Bernardi, Luca Magazzini, Luciano Tadiello, Valeria Cipolletti, Marco Arimondi.
      Discovering the non-isoprene components retained within the natural rubber (NR) matrix is essential to elucidate their role in modulating the viscoelastic properties of the raw material and the rheological behavior of the rubber-based materials, such as tyres in the automotive industry. Here, lipid's content, their identification and quantification were achieved by hydrophilic interaction liquid chromatography (HILIC) coupled to Fourier-transform (FT) orbital-trap tandem mass spectrometry (MS/MS) through electrospray ionization (ESI). Overall, 10 classes of phospholipids (PLs) and a few glycolipids (GLs) were characterized also in terms of regiochemistry including 7 species of fatty acids (FAs), 107 PLs and 6 GLs. Among the identified PLs, phosphatidylcholines (PCs), phosphatidylinositols (PIs), phosphatidylethanolamines (PEs) were detected, with a marked predominance of their lyso-forms, including lyso-N-acyl phosphatidylethanolamines (LNAPEs), lyso-phosphatidylserines (LPSs), and lyso-phosphatidic acids (LPAs). In addition, the presence of digalactosyl monoacylglycerols (DGMGs) was established. The identified FA species predominantly contained even numbers of carbon atoms, mainly C16 and C18, with varying degrees of unsaturation. By comparing various types of NR in terms of geographical origin (Thailand, Cameroon, Brazil) and different raw material treatment (acid, neutral and basic), a dissimilar content of lipids was reflected across the different sample groups.
    Keywords:  Fatty acids; GC-FID; Glycolipids; LC-ESI-Tandem MS; Natural rubber; Phospholipids
    DOI:  https://doi.org/10.1016/j.talanta.2025.129150
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