bims-metlip 2025-11-02 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–11–02
29 papers selected by
Sofia Costa, Matterworks

Trapped Ion Mobility Improves Annotation Accuracy in LC-HRMS Screening Applications for Exposomics.
Adaptive Modeling of Tandem Mass Spectrometry Data: Creation of the METLIN 960K MRM Database.
LC-MS/MS Method Development and Validation for Clinical Pharmacokinetics and Therapeutic Drug Monitoring of Potassium-Competitive Acid Blocker Vonoprazan-Based Triple Therapy for H. pylori in Human Plasma.
Enhanced Spatial Proteomics and Metabolomics from a Single Tissue Section Using MALDI-MSI and LCM-microPOTS Platforms.
Investigating the Quantitative Structure-Ionization Efficiency Relationship of Small Molecules and Lipids in the Presence of Ammonium Fluoride in MALDI-TIMS-QTOF Mass Spectrometry Imaging.
Comparative analysis of LC-MS/MS and electrochemical immunosensor for clarithromycin detection in plasma.
Entecavir HPLC-MS/MS quantitation and optimized sample preparation using human plasma: Validation in a pharmacokinetic study.
Analytical Methods for the Determination of Diamorphine (Heroin) in Biological Matrices: A Review.
An untargeted data mining strategy for extracting chemical exposome signatures from LC-HRMS data: Application to meconium for early-life exposure assessment.
Identifying Novel DNA Adducts in Amphipods and Developing Sample Preparation for Adductomics Using Dispersive Solid-Phase Extraction.
Aerith: Visualization and Annotation of Isotopic Enrichment Patterns of Peptides and Metabolites with Stable Isotope Labeling from Proteomics and Metabolomics.
Metabolic Impact of Pesticides on Radish: 2D and 3D Mass Spectrometry Imaging of Metabolites in Raphanus sativus.
Measurement of twenty-one serum steroid profiles by UPLC-MS/MS for the diagnosis and monitoring of congenital adrenal hyperplasia.
Optimised reverse-phase chromatography for clinical detection of vitamins A and E in serum.
Enhanced LC-MS Detection of γ-Aminobutyric Acid via Trimethylation Enhancement Using Diazomethane.
Comprehensive lipid analysis of human meibum and tears.
Single phase extraction method for determination of dithianon residues in fruits and vegetables using LC ESI (-) MS/MS.
Quantitative Targeted Analysis of Antibody Fc Glycosylation by Glyco-MRM.
MSInet: A Self-Supervised CNN Framework Integrating Global and Local Context for Robust Mass Spectrometry Imaging Segmentation.
An Innovative Use of the QuEChERs Method and LC-MS/MS Technique for Fast and Simple Determination of Quinolizidine Alkaloids in Leguminous Plants.
Validated LC-MS/MS method for cisplatin quantification in plasma, whole blood, and cervical cancer tissue via diethyldithiocarbamate derivatization: Clinical application in cervical cancer.
In Vitro Metabolite Profiling of 4,4-Diaminodiphenylmethane Using Human Liver S9 Fractions and LC-Orbitrap HRMS: In Silico Toxicity Studies of Its Metabolites.
Screening of biomarkers for early diagnosis of trauma-induced coagulopathy based on untargeted metabolomics.
High-Frequency Microfluidic Fractionation for Compound-Resolved Bioactivity-Based Metabolomics.
Cell Metabolomics Reveals the Hepatotoxic Mechanism of Copper in Normal Rat Liver Cells Using Reversed-Phase and Hydrophilic Interaction Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.
AI-SNPS2: A Multi-Layered LC-MS/MS Platform Integrating Molecular Networking and Retention Time Prediction for Identifying Controlled and New Psychoactive Substances.
Barcode-free hit discovery from massive libraries enabled by automated small molecule structure annotation.
Analysis of growth hormone releasing hormone and its analogs in urine using nano liquid chromatography coupled with quadrupole/orbitrap mass spectrometry.
Development and Modification of Ion Mobility Mass Spectrometry Instrumentation for Enhanced Analysis of Biological Molecules.

Anal Chem. 2025 Oct 31.

Trapped Ion Mobility Improves Annotation Accuracy in LC-HRMS Screening Applications for Exposomics.

Carolin Huber, Nadin Ulrich, Martin Krauss.

Ion mobility techniques coupled to mass spectrometry, such as trapped ion mobility (TIMS), are promoted to separate analytes from coeluting matrix interferences and to resolve isomers based on their corresponding CCS values. Complementary to the retention time (RT) dimension revealed from liquid chromatography, the collision cross section (CCS) serves as a robust and matrix-independent parameter. We evaluated the advantages of TIMS in the screening of human samples, such as urine, serum, breastmilk, and matrices relevant for exposure analysis, such as dust and wastewater. We conducted a screening library for 769 environmental contaminants, which resulted in a total of 948 CCS values (594 positive and 354 negative ionization modes). We screened for the potential co-occurrence of interfering compounds originating from five different matrix backgrounds, leading to peaks with similar m/z and RT but differences in the mobilograms. For all matrices combined, 112 peaks with different mobility values relative to the reference standard were found. Our evaluation highlights the benefits of TIMS in reducing the number of inconclusive assignments through the separation of coeluting compounds and background noise and gaining a high MS2 coverage for low-abundant ions. These advantages are beneficial especially for suspect screening applications, where broader RT windows are necessary.

DOI: https://doi.org/10.1021/acs.analchem.5c04665
Anal Chem. 2025 Oct 28.

Adaptive Modeling of Tandem Mass Spectrometry Data: Creation of the METLIN 960K MRM Database.

Aries Aisporna, Bill Webb, Winnie Uritboonthai, Linh Hoang, Elizabeth M Billings, Corey Hoang, Robert Plumb, Gary Siuzdak.

Multiple Reaction Monitoring (MRM) remains the gold standard for quantitative mass spectrometry but continues to be constrained by the limited availability of high-quality transitions and collision energy (CE) values for many biologically and chemically relevant molecules. Here, we present the METLIN 960K MRM library, a 960,000-compound transition resource derived entirely from empirically acquired MS/MS data. MRM transitions were generated in both positive and negative ionization modes using an empirical spline-based pipeline refined by AI BioSync, an XCMS enhancement that provides a framework of AI and machine-learning tools designed to decipher spectral data for biological and analytical relevance. Central to this approach is spline fitting of CE-dependent intensity profiles from experimental MS/MS data collected at four discrete energies (0, 10, 20, and 40 eV), enabling continuous CE modeling and precise prediction of optimal fragmentation conditions. Supervised learning models were used within AI BioSync to refine spline fitting across diverse chemical classes, improving reproducibility and predictive accuracy. Validation across more than 100 authentic compounds, including rare metabolites and diverse small molecules, demonstrated robust detection down to 1 nM, confirming both sensitivity and scalability. This framework also holds immediate applicability for preclinical drug development studies, where authentic metabolite and impurity standards are often unavailable. Unlike prior methods reliant on in silico fragmentation or heuristic rules, all transitions are derived directly from experimental MS/MS data using absolute intensities. The resulting precursor m/z-centric METLIN 960K MRM library (https://metlin.scripps.edu) greatly expands the chemical space accessible to targeted quantitation, providing a scalable, vendor-independent path for sensitive and specific molecular detection across research, clinical, and applied applications.

DOI: https://doi.org/10.1021/acs.analchem.5c04639
Pharmaceuticals (Basel). 2025 Oct 08. pii: 1509. [Epub ahead of print]18(10):

LC-MS/MS Method Development and Validation for Clinical Pharmacokinetics and Therapeutic Drug Monitoring of Potassium-Competitive Acid Blocker Vonoprazan-Based Triple Therapy for H. pylori in Human Plasma.

Naser F Al-Tannak, Hani M Hafez, Ahmed Hemdan, Abdullah Aldasem, Ibrahim Khadra.

  Background: A novel triple therapy regimen for Helicobacter pylori eradication, recently approved by the U.S. FDA, comprises vonoprazan (VPN), a potassium-competitive acid blocker, in combination with amoxicillin (AMX) and clarithromycin (CMN). This study presents the development and full validation of a rapid, selective, and sensitive LC-MS/MS method for the simultaneous quantification of these three drugs in spiked human plasma. Methods: Sample preparation was performed using a simple and efficient liquid-liquid extraction (LLE) technique. Chromatographic separation was achieved within 5 min using a Phenomenex Kinetex C18 column (100 × 4.6 mm, 2.6 µm) and a gradient elution system consisting of 0.1% formic acid in water and acetonitrile. Moreover, diazepam was used as an internal standard. The mass spectrometric detection was conducted in multiple reaction monitoring (MRM) mode using positive electrospray ionization. Results: The method exhibited excellent linearity over the investigated concentration ranges (2, 5, 10, 20, 50, and 100 ng/mL for amoxicillin and clarithromycin and 5, 10, 20, 30, 50, and 100 ng/mL for vonoprazan). Intra- and inter-day precision and accuracy values met FDA bioanalytical method validation guidelines, with relative standard deviations and relative errors below 15%. Mean absolute recoveries were above 93% for all analytes. Conclusions: The developed method was fully validated, rapid, selective, and sensitive LC-MS/MS and was assessed using the AGREE tool as a greenness assessment approach, confirming its environmental friendliness and alignment with green analytical chemistry principles.

Keywords:  H. pylori infection; LC-MS/MS; amoxicillin; clarithromycin; green chemistry; vonoprazan

DOI:  https://doi.org/10.3390/ph18101509
Anal Chem. 2025 Oct 31.

Enhanced Spatial Proteomics and Metabolomics from a Single Tissue Section Using MALDI-MSI and LCM-microPOTS Platforms.

Marija Veličković, Le Z Day, Kevin J Zemaitis, Isaac Kwame Attah, Kristin E Burnum-Johnson, Christopher R Anderton, Dušan Veličković.

Spatially resolved mass spectrometry (MS)-based multiomics workflows are becoming more utilized for revealing the complex biology that occurs within tissues. However, these approaches commonly require multiple independent tissue sections to analyze the metabolite and protein compositions of these samples. This poses a significant challenge in preserving cell- or region-specific molecular fidelity, as variations between tissue sections can compromise the accurate correlation of molecular data. Here, we developed workflows for comprehensive multiomics profiling from a single tissue section (STS) using different MS modalities. We enhanced the functionality of an electrically insulated substrate by employing metal-assisted approaches that enabled both MS-based untargeted spatial metabolomics and proteomics from STS. This allowed metabolite imaging using matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI), without compromising it for subsequent proteome profiling with laser capture microdissection (LCM)-based technology. Specifically, implementing copper tape as a backing for polyethylene naphthalate (PEN) slides enabled the detection of >140 metabolites across a poplar root tissue section using MALDI-trapped ion mobility spectrometry time-of-flight (timsTOF)-MS. Afterward, we detected 6571 unique proteins from two distinct root regions by leveraging LCM technology coupled to our microdroplet based sample preparation approach. We also developed an alternative workflow utilizing gold-coated PEN substrates for imaging with MALDI-Fourier-transform ion cyclotron resonance (FTICR)-MS, which permitted the profiling of >170 metabolites and the identification of 6542 unique proteins across a single poplar root tissue section. These results were comparable to using each omics analysis independently. These approaches offer new opportunities for high-resolution molecular profiling of multiple omics levels across biological tissues.

DOI: https://doi.org/10.1021/acs.analchem.5c05005
J Am Soc Mass Spectrom. 2025 Oct 28.

Investigating the Quantitative Structure-Ionization Efficiency Relationship of Small Molecules and Lipids in the Presence of Ammonium Fluoride in MALDI-TIMS-QTOF Mass Spectrometry Imaging.

Varvara Nikolopoulou, Arthur Stem, Vasilis Vasiliou, Reza Aalizadeh.

Metabolites are essential small molecules that are naturally occurring in biological processes as end or intermediate products of various pathways. Matrix-assisted laser desorption/ionization-trapped ion mobility separation-mass spectrometry imaging (MALDI-TIMS-MSI) is an emerging technique that can be used to identify the spatial localization of endogenous compounds on tissue. We evaluated the potential of ammonium fluoride (NH4F) to enhance the ionization efficiency of metabolites in negative polarity mode when used as a comatrix additive in N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC), 9-aminoacridine (9AA), and 1,5-diaminonaphthalene (DAN) matrices. An extensive list of 234 isotopically labeled metabolites (IROA-IS) was used to establish a quantitative ionization efficiency model with respect to the metabolite chemical structures. In addition, we extended our evaluation to endogenous compounds observed in brain samples collected from male mice. Overall, our study demonstrates that NH4F improves the sensitivity and ionization efficiency of metabolites and lipids in MALDI-TIMS-MSI. This effect was found to vary depending on the matrix, with the ionization efficiency of the studied metabolites increasing in the order NEDC < 9AA < DAN. The quantitative structure-ionization efficiency relationship model can facilitate the appropriate selection of the matrix in MALDI prior to the analysis of analytes of interest.

DOI: https://doi.org/10.1021/jasms.5c00211
Mikrochim Acta. 2025 Oct 28. 192(11): 758

Comparative analysis of LC-MS/MS and electrochemical immunosensor for clarithromycin detection in plasma.

Mohamed Zouari, Ahmet Cetinkaya, Sibel A Ozkan.

  The development and comparison of two distinct analytical approaches is presented for quantifying clarithromycin in human plasma: a method based on ultra-performance liquid chromatography coupled with tandem mass spectrometry and a novel electrochemical immunosensor. The use of both techniques is justified by their ability to meet distinct operational requirements, ranging from high-throughput centralized analysis to rapid, point-of-care monitoring, which is essential for therapeutic drug monitoring and pharmacokinetic studies. The developed chromatographic method, which combines protein precipitation with subsequent liquid-liquid extraction using tert-butyl methyl ether, demonstrated robust and reproducible performance. Demonstrating excellent linearity and precision within the validated concentration range (0.1 to 4.0 µg per milliliter), the method achieved a limit of detection as low as 0.03 µg ml-1. In parallel, an electrochemical immunosensor was developed using functionalized magnetic beads and screen-printed carbon electrodes. The biosensor demonstrated the same detection limit as the chromatographic method, a rapid analysis time of under 30 min, and high selectivity even in complex biological matrices. By comparing these two platforms, the study highlights their complementary strengths: the robustness and regulatory alignment of liquid chromatography-mass spectrometry versus the portability, speed, and operational simplicity of the immunosensor. This dual approach offers a more flexible and context-specific strategy for clarithromycin monitoring, from centralized laboratory workflows to decentralized or point-of-care applications.

Keywords:  Bioanalytical comparison; Clarithromycin; Functionalized magnetic beads; Dual-platform strategy; Electrochemical immunosensor; Human plasma; Liquid chromatography-tandem mass spectrometry; Screen-printed carbon electrode

DOI:  https://doi.org/10.1007/s00604-025-07652-6
Int J Clin Pharmacol Ther. 2025 Oct 28.

Entecavir HPLC-MS/MS quantitation and optimized sample preparation using human plasma: Validation in a pharmacokinetic study.

Rui Zhao, Pengfei Li, Hongchuan Liu, Ping Du, Lihong Liu.

AIMS: To determine the entecavir levels in human plasma using a quantitative high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) technique.
MATERIALS AND METHODS: The developed method employed isotope-labeled internal standard, a high-throughput solid phase extraction process for sample preparation, and HPLC-MS/MS analysis using multiple reaction monitoring transitions in positive mode.
RESULTS: With a 0.025 - 10 ng/mL linear range, interday and intraday accuracy ranged from -3.4 to 5.3%; between-day and within-day precision was ≤ 7.3%. Stability, matrix effect, and recovery were all well within the acceptable criteria. A pharmacokinetic study of entecavir dispersible tablets at an oral dosage of 0.5 mg was successfully carried out using the validated method.
CONCLUSION: The established HPLC-MS/MS method proved effective for quantitation of entecavir in human plasma.

DOI: https://doi.org/10.5414/CP204582
Toxics. 2025 Oct 13. pii: 867. [Epub ahead of print]13(10):

Analytical Methods for the Determination of Diamorphine (Heroin) in Biological Matrices: A Review.

Ahmed Ibrahim Al-Asmari.

  Diamorphine (DIM, heroin) is a semi-synthetic opioid that undergoes rapid conversion to 6-monoacetylmorphine and morphine, producing short-lived biomarkers that are difficult to capture during the process. This review critically explores the evolution of analytical techniques for quantitative DIM analysis in biological matrices from 1980 to 2025. It synthesizes findings across blood, plasma, urine, hair, sweat, and postmortem samples, emphasizing matrix-specific challenges and forensic applicability. Unlike previous opioid reviews that primarily focused on metabolites, this work highlights analytical methods capable of successfully detecting diamorphine itself alongside its key metabolites. This review examines 32 studies spanning three decades and compares three core analytical methods: gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC) with optical detection and liquid chromatography-mass spectrometry (LC-MS). Key performance metrics include sensitivity, sample preparation workflow, hydrolysis control, metabolite coverage, matrix compatibility, automation potential and throughput. GC-MS remains the workhorse for hair and sweat ultra-trace screening after derivatization. HPLC with UV, fluorescence or diode-array detection enables robust quantification of morphine and its glucuronides in pharmacokinetic and clinical settings. LC-MS facilitates the multiplexed analysis of DIM, its ester metabolites and its conjugates in a single, rapid run under gentle conditions to prevent ex vivo degradation. Recent advances such as high-resolution mass spectrometry and microsampling techniques offer new opportunities for sensitive and matrix-adapted analysis. By integrating validation parameters, forensic applicability, and evolving instrumentation, this review provides a practical roadmap for toxicologists and analysts navigating complex biological evidence.

Keywords:  forensics; metabolites; opiates; opioids; pharmacokinetics; toxicology

DOI:  https://doi.org/10.3390/toxics13100867
Anal Chim Acta. 2025 Dec 15. pii: S0003-2670(25)01145-6. [Epub ahead of print]1379 344751

An untargeted data mining strategy for extracting chemical exposome signatures from LC-HRMS data: Application to meconium for early-life exposure assessment.

Dylan Saunier, Éric Venot, Blanche Guillon, Sylvain Dechaumet, Florence Castelli, Etienne Thevenot, François Fenaille, Blandine de Lauzon-Guillain, Karine Adel-Patient, Estelle Rathahao-Paris.

   BACKGROUND: Exposome research has expanded rapidly in recent years, driven by advances in analytical techniques such as liquid chromatography-high-resolution mass spectrometry (LC-HRMS), which enable broad and sensitive chemical coverage. Targeted methods focus on known compounds, while untargeted metabolomic approaches provide a more holistic view and may reveal exposure biomarkers, but they are not specifically designed to detect exogenous chemicals. Identifying relevant exposure markers within the vast and complex datasets generated by untargeted LC-HRMS data remains a significant analytical and computational challenge, requiring innovative data mining strategies.
RESULTS: We developed a novel untargeted data mining strategy to extract exogenous chemical signatures from complex LC-HRMS datasets. The approach integrates isotopic signature enrichment (ISE), biotransformation-informed feature selection and an "exposure rate" metric. When applied to meconium data from the EDEN cohort, the strategy led to a six-fold reduction in the number of features by retaining only those exhibiting valid carbon isotope patterns. Mass defect plots revealed signatures of suspect monohalogenated species and putative conjugated and non-conjugated metabolites in a specific region. Incorporating ISE results into the chemical formula prediction significantly reduced the number of candidates, improving annotation efficiency. In utero exposure to xenobiotics was supported by the detection of known exposure markers such as acetaminophen, caffeine and nicotine. These results demonstrate the method's potential to uncover exposomic signals in complex biological matrices.
SIGNIFICANCE: This study presents a novel data mining strategy that reduces the complexity of untargeted LC-HRMS data by retaining chemically reliable features based on isotopic signatures. As a proof of concept, this strategy enables the detection of specific chemical signatures and exogenous compounds without prior knowledge. Its adaptability to various biological matrices and its compatibility with different high-resolution mass spectrometry platforms make this strategy a valuable tool for exposome research and early-life exposure assessment.

Keywords:  Chemical exposome; Early life exposure; Large LC-HRMS datasets; Mass defect profile; Untargeted data mining

DOI:  https://doi.org/10.1016/j.aca.2025.344751
Environ Sci Technol. 2025 Oct 30.

Identifying Novel DNA Adducts in Amphipods and Developing Sample Preparation for Adductomics Using Dispersive Solid-Phase Extraction.

Zareen Khan, Elena Gorokhova, Giulia Martella, Nisha H Motwani, Natalia Tretyakova, Pedro F M Sousa, Hitesh V Motwani.

  The growing use of environmental DNA adductomics for exposure assessment emphasizes the need for improved methods that are adapted to chemically and biologically diverse samples. While liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) has advanced DNA adductome analysis, sample cleanup methods remain challenging, especially in nonmammalian models. Here, we used amphipod Monoporiea affinis, a sentinel species in biological effect studies, as a surrogate crustacean species for the method development. It is particularly suitable because its chitinous tissues and high lipid content present a challenging matrix for DNA extraction. We addressed the following objectives: (i) to propose adduct structures by combining multiple tools for DNA adduct screening and (ii) to evaluate the applicability of dispersive solid-phase extraction (d-SPE) as a cleanup step for DNA adductome analysis. Toward the first objective, we integrated open-source software nLossFinder with a recently introduced DNA adductomics database (GitLab) to enhance the structural identification of unknown adducts. A combination of accurate mass data and MS2-fragmentation allowed differentiation of the complex mixture of nucleoside adducts, facilitating the structural identification of 16 DNA adducts, including 10 modifications on amphipod DNA reported for the first time. Toward the second objective, we introduced d-SPE as a novel cleanup approach for DNA adduct analysis in whole-body crustacean samples. Using Z-sep+ as the d-SPE sorbent, we demonstrated a major reduction of matrix interferences, including phospholipids, and enhanced sensitivity toward DNA adducts detection, leading to improved LC-HRMS signal response by up to 170%. d-SPE offers a simple alternative to conventional methods like standard SPE and liquid-liquid extraction, making it a valuable tool for DNA adductomics in environmental monitoring and aiding high-throughput capacity, especially while handling large numbers of samples. Further studies should include validation of this method for other species and DNA modifications. These advancements underscore the potential of the proposed data analysis workflow and d-SPE for improving mass spectrometry-based DNA adductomics in environmental monitoring, paving the way for more accurate and comprehensive exposure assessments across diverse species and environmental conditions.

Keywords:  DNA adducts; dispersive solid-phase extraction; exposure assessment; high-resolution mass spectrometry; nontarget analysis

DOI:  https://doi.org/10.1021/acs.est.5c02493
Anal Chem. 2025 Oct 28.

Aerith: Visualization and Annotation of Isotopic Enrichment Patterns of Peptides and Metabolites with Stable Isotope Labeling from Proteomics and Metabolomics.

Yi Xiong, Ryan S Mueller, Shichao Feng, Xuan Guo, Chongle Pan.

Stable isotope probing (SIP) traces the metabolism of biological cells using isotopically heavy substrates (e.g., 13C, 15N, or 2H). Confident identification of the metabolic products of isotopic labeling remains a challenge due to the difficulties in simulating, visualizing and annotating the isotopic patterns of partially labeled peptides and metabolites found in mass spectrometry (MS) data. Here, we present Aerith, an R package designed to visualize data of simulated and observed isotopic envelopes of peptides and metabolites with user-defined formula and atom % enrichment levels. Aerith models the isotopic distributions of the fragment ion series of a peptide by sequentially convoluting isotopic envelopes of monomeric units using a convolution algorithm. Aerith simulates fine isotopic structures of a compound using Monte Carlo simulation via the multinomial distribution, and calculates the isotopic envelopes of metabolites with known chemical formulas using an FFT-based algorithm. These algorithms provide accurate simulation of the isotopic envelopes of SIP-labeled peptides and metabolites with high computational efficiency. Aerith evaluates peptide-spectrum matches through multiple robust and commonly used scoring functions to compare experimental and theoretical spectra. These algorithms were implemented in C++ and accessed in R via Rcpp to ensure real-time interactivity and significantly improve computational efficiency compared to native R code. We present case studies to demonstrate Aerith's utility in resolving isotopic fine structures and envelopes for glucose, penicillin, and microbial peptides containing natural and enriched isotopes. By providing visualization of isotopically labeled peptides and metabolites, Aerith enables precise annotation of their mass spectra and manual validation of their identifications in proteomic and metabolomic SIP studies.

DOI: https://doi.org/10.1021/acs.analchem.5c03207
J Agric Food Chem. 2025 Oct 29.

Metabolic Impact of Pesticides on Radish: 2D and 3D Mass Spectrometry Imaging of Metabolites in Raphanus sativus.

Sumi Krupa, Tomasz Ruman, Joanna Nizioł.

  Pesticides are essential for crop protection but can markedly reshape plant metabolism with implications for food quality and safety. This work introduces an integrated strategy that combines untargeted ultrahigh-performance liquid chromatography-ultrahigh-resolution mass spectrometry (UHPLC-UHRMS) with two- and three-dimensional mass spectrometry imaging using laser ablation remote atmospheric pressure photoionization/chemical ionization (LARAPPI/CI-2D/3D-MSI) to elucidate global and spatial metabolic responses of radish to pesticide exposure. The results reveal compound- and dose-dependent effects: field-relevant concentrations cause minor metabolic perturbations, whereas 100-fold higher doses induce systemic reprogramming of amino acid, carbohydrate, lipid, and secondary metabolism. MSI uncovers distinct tissue- and depth-specific patterns of metabolic alteration, demonstrating nonadditive responses to pesticide mixtures. By linking molecular profiling with spatial metabolite mapping, this work advances the mechanistic understanding of plant stress responses and provides a framework for evaluating the metabolic consequences of pesticide regimes on crop physiology and food safety.

Keywords:  UHPLC-UHRMS; environmental stress response; laser ablation; photoionization; plant metabolomics; untargeted metabolomics

DOI:  https://doi.org/10.1021/acs.jafc.5c12445
J Mass Spectrom Adv Clin Lab. 2025 Dec;38 10-17

Measurement of twenty-one serum steroid profiles by UPLC-MS/MS for the diagnosis and monitoring of congenital adrenal hyperplasia.

Joon Hee Lee, Kyunghoon Lee, Sun-Hee Jun, Yun Jeong Lee, Choong Ho Shin, Young Ah Lee, Junghan Song.

   Background: Congenital adrenal hyperplasia (CAH) represents a group of inherited disorders affecting steroidogenesis. Early and accurate diagnosis is crucial for effective treatment, particularly for preventing adrenal insufficiency and minimizing androgen excess. This study aims to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of 21 steroid hormones, including 11-oxygenated androgens, which are critical for diagnosing and monitoring various forms of CAH.
Methods: We utilized a microbore column UPLC combined with hydroxylamine derivatization, which enabled excellent chromatographic separation and enhanced sensitivity of all target compounds. The method was evaluated for precision, linearity, recovery, ion suppression, and carryover according to FDA and CLSI guidelines. Steroid profiles from healthy controls and CAH patients were compared using Mann-Whitney tests.
Results: The UPLC-MS/MS method demonstrated excellent precision (<20 % except for 11-ketoandrostenedione), linearity (R 2 > 0.99), low limits of detection and quantification, and satisfactory recovery (57-86 % absolute, 99-111 % relative). Our method showed good correlation with proficiency testing group means, although significant negative biases were noted for androstenedione, progesterone, and 11-deoxycortisol. In a clinical setting, significant increases in pregnenolone, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and other key steroids were observed in patients with 21-hydroxylase deficiency, while distinct profiles were identified for patients with 17-hydroxylase deficiency, cytochrome P450 oxidoreductase deficiency, and lipoid CAH.
Conclusions: Our UPLC-MS/MS method provides a sensitive and specific tool for the comprehensive profiling of adrenal steroids, offering improved diagnostic accuracy for CAH. Its ability to differentiate between various CAH subtypes highlights its potential clinical utility in both diagnosis and monitoring.

Keywords:  Congenital adrenal hyperplasia; Inborn errors of metabolism; Method validation; Steroids; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jmsacl.2025.10.003
Sci Rep. 2025 Oct 29. 15(1): 37814

Optimised reverse-phase chromatography for clinical detection of vitamins A and E in serum.

Aliya Annujma Shahrunizam, Siok-Fong Chin, Noraidatulakma Abdullah, Azmawati Mohamed Nawi, Rahman Jamal.

  Vitamin A and E are indispensable fat-soluble vitamins that play pivotal roles in promoting human health and well-being. Accurate quantification of these vitamins in biological samples is critical. Thus, we aim to develop and optimise a sensitive method suitable for simultaneous analysis of vitamin A and E. We introduced a method for simultaneous detection of Vitamin A and E using reverse-phase high-performance liquid chromatography (RP-HPLC). Calibration curves and quality control samples were prepared using a 4% of Bovine Serum Albumin (BSA) matrix. The analytes and internal standard (ISTD), Dodecanophenone were effectively separated using a polar column and detected via UV detector at wavelengths of 255 nm, 298 nm, and 325 nm respectively, with a total run time of 30 min. Results: The method demonstrated excellent linearity with regression coefficients (r²) > 0.995. The optimisation process of the HPLC method affirmed its precision and reliability, meeting analytical chemistry standards. Subsequently, this validated method was applied to serum samples obtained from patients and a population cohort exhibiting distinct clinical pathologies, including individuals without colorectal cancer (CRC), those with symptomatic and asymptomatic CRC. Conclusions: In conclusion, this HPLC method offers a reliable means for routine quantification of vitamin A and E in human serum, holding promise for enhancing nutritional research and clinical diagnostics. Its novelty lies in the use of a surrogate BSA matrix with a polar RP-HPLC column and multi-wavelength detection, enabling robust quantification of endogenous analytes in clinically relevant cohorts.

Keywords:  Optimisation; Serum; UHPLC; Vitamin A; Vitamin E

DOI:  https://doi.org/10.1038/s41598-025-21652-9
J Am Soc Mass Spectrom. 2025 Oct 30.

Enhanced LC-MS Detection of γ-Aminobutyric Acid via Trimethylation Enhancement Using Diazomethane.

Christian A Rosales, Noah A Lepinsky, Mohamed Ramadan, Jeffrey M Manthorpe, Michael G Jonz, Jeffrey C Smith.

  Neurotransmitters are critical for the proper function, signal transmission, and physiological balance of the brain, with γ-aminobutyric acid (GABA) being the main inhibitory neurotransmitter in the central nervous system. GABA is present at relatively low concentrations compared to other neurotransmitters, therefore requiring sensitive analytical methods for accurate identification and quantitation. Described herein is a rapid and facile liquid chromatography mass spectrometry (LC-MS)-based chemical derivatization method to enhance the detection of GABA, demonstrated in both saline culture media and Carassius auratus (goldfish) retina samples. We have expanded the use of trimethylation enhancement using diazomethane (TrEnDi) to permethylate GABA ([GABATr]+) at 98-100% yields across all matrix types. Quantitative methylation of the carboxylic acid and amino moieties nullifies any zwitterionic character and fixes a permanent positive charge on [GABATr]+, leading to MS sensitivity enhancement. In biological triplicates of goldfish retina samples, [GABATr]+ boasted 6.3-27.9-fold increases in MS sensitivity compared to its unmodified counterpart, enabling quantitation with concentrations ranging between 78.6 and 806.5 nM. Calibration curve linearity for [GABATr]+ and unmodified GABA was R2 = 0.9996 and R2 = 0.9923, respectively. Limits of detection and quantitation (LOD/LOQ) for [GABATr]+ were 0.053 nM (1.1 fmol)/0.18 nM (3.6 fmol), compared to 2.5 nM (50 fmol)/8.3 nM (167 fmol) for unmodified GABA. This work demonstrates that TrEnDi has the ability to rapidly enhance LC-MS detection of GABA in a relatively facile manner, reducing the probability of reporting false negatives in the analysis of complex biological samples.

Keywords:  Carassius auratus; HPLC; TrEnDi; diazomethane; mass spectrometry; neurotransmitter; γ-aminobutyric acid (GABA)

DOI:  https://doi.org/10.1021/jasms.5c00294
Sci Rep. 2025 Oct 30. 15(1): 35556

Comprehensive lipid analysis of human meibum and tears.

Makiko Terai, Keisuke Jojima, Megumi Ogawa-Sawai, Akio Kihara.

  Abnormalities in the tear film lipid layer, which plays a critical role in preventing water evaporation and protecting the corneal surface, lead to dry eye disease. The lipids in this layer include both meibum lipids (from the meibomian glands) and phospholipids of other origins. Meibum lipids include cholesteryl esters, wax monoesters, wax diesters (WdiEs), (O-acyl)-ω-hydroxy fatty acids (OAHFAs), and cholesteryl OAHFAs. Nonetheless, the exact composition of these lipid classes remains largely unclear. Here, we analyze the composition of cholesteryl esters, wax monoesters, WdiEs, OAHFAs, cholesteryl OAHFAs, phosphatidylcholines, and sphingomyelins in human meibum and tears using multiple reaction monitoring mode liquid chromatography-tandem mass spectrometry, which is highly sensitive, selective, and quantitative. This revealed that the WdiEs in meibum and tears fall within the type 1ω and 2ω classes. Among the lipids examined, the type 1ω WdiEs in particular comprised diverse species. The lipid composition of most of the lipid classes, except for the phosphatidylcholines, was similar in meibum and tears. The findings of this comprehensive lipid analysis contribute to elucidating the overall composition of human meibum and tear lipids.

Keywords:  Meibum; Multiple reaction monitoring; Tandem mass spectrometry; Tear; Tear film lipid layer; Wax diester

DOI:  https://doi.org/10.1038/s41598-025-23048-1
Sci Rep. 2025 Oct 31. 15(1): 38112

Single phase extraction method for determination of dithianon residues in fruits and vegetables using LC ESI (-) MS/MS.

Esmail Elsayed Aboshanab, Mahmoud Hamdy Abdelwahed, Sanaa A M El-Sawi, Mostafa M Emara, Ahmed A Omran.

  Dithianon is a non-systemic fungicide, applied in some agricultural products. Dithianon residues in food cause health problems for humans so it is recommended to be analyzed in fruits and vegetables. Four different extracting solvents were compared to get the optimum one. Quantitative analysis was done using a liquid chromatography triple quadruple mass spectrometer in different agricultural products. The in-house validation process was carried out based on SANTE guideline. The results demonstrated an average recovery rate between 85 and 113%, with relative standard deviations (RSDs) ≤ 8% for all tested food matrices in repeatability and RSDwR% = 16% in within-Laboratory reproducibility. Good linearity at r2 > 0.99 was obtained for 0.001-0.5 µg/ml dithianon calibration curves. Limit of quantifications (LOQs) for the method ranged between 0.01 and 0.05 µg/g with expanded measured uncertainty Uexp = ± 42%. Our method is simple, fast and reliable for the determination of dithianon residues in food so, it is recommended to be applied in the routine analysis. The method's practicality was confirmed by analyzing fifty market samples from Egypt. No dithianon residues were detected, a finding consistent with its limited national registration and underscoring the method's utility for ensuring compliance and food safety.

Keywords:  Dithianon; Fruits; LC-MS/MS; Pesticides residues; Validation; Vegetables

DOI:  https://doi.org/10.1038/s41598-025-23528-4
J Proteome Res. 2025 Oct 28.

Quantitative Targeted Analysis of Antibody Fc Glycosylation by Glyco-MRM.

Yue Ma, Yang Song, Ji Luo, Jingxia Wang, Yi-Wei Tang, Dapeng Zhou.

  The N-glycoforms of Fc domain critically regulate binding affinity of IgG1 to Fcγ receptor IIIA (FcγRIIIa), Fcγ receptor IIb (FcγRIIb), and complement components. Quantifying antigen-specific IgG1 glycopeptides may provide precise insights into the pathogenesis of severe viral infections and autoantibody-mediated diseases. Here, we developed a liquid chromatography (LC) coupled with mass spectrometry (MS) by the multiple reaction monitoring (MRM) method to analyze IgG1 glycosylation profiles. Calibration curves were generated for six glycopeptides with integrated isotope-labeled internal standards, yielding lower limits of quantification (LLOQ) of G2 (200 pg/mL, 70.92 pM), G0F (500 pg/mL, 189.39 pM), G0NF (40 pg/mL, 14.05 pM), G2S (2.5 ng/mL, 802.87 pM), G1 (500 pg/mL, 187.98 pM), and G1N (100 pg/mL, 34.93 pM). For absolute IgG1 quantification, the LLOQ was determined as 1.26 μg/mL (8.43 nM). Application of calibration curve-based assays to influenza and COVID-19 infected individuals (within 3 months after infection) revealed distinct glycosylation profiles: influenza infected individuals exhibited significantly reduced core-fucosylation (28%), while both disease groups showed elevated galactosylation levels. This methodology provides a platform for laboratory-developed tests to track glycosylation alterations using widely accessible liquid chromatography-mass spectrometry (LC-MS) equipment.

Keywords:  B4GalTs; FUT8; Fc glycosylation; IgG1; MRM; mass spectrometry

DOI:  https://doi.org/10.1021/acs.jproteome.5c00560
Anal Chem. 2025 Oct 28.

MSInet: A Self-Supervised CNN Framework Integrating Global and Local Context for Robust Mass Spectrometry Imaging Segmentation.

Mudassir Shah, Siyang Liu, Lei Guo, Yusong Zhang, Lingli Deng, Xiangnan Xu, Zhaodong Fei, Jingjing Xu, Jiyang Dong.

Mass spectrometry imaging (MSI) enables label-free molecular mapping in tissues but presents challenges for spatial segmentation due to high dimensionality, nonlinear spectral variation, and tissue heterogeneity. Traditional unsupervised clustering methods often rely on predefined cluster numbers and overlook spatial information, yielding fragmented or biologically implausible results. We introduce MSInet, a self-supervised deep learning framework for robust, annotation-free MSI segmentation. MSInet combines two strategies within a convolutional neural network: patch-wise contrastive learning to capture global semantic relationships, and superpixel-guided refinement to enforce local spatial consistency. This dual-consistency design simultaneously enhances global context awareness and local boundary precision during training. MSInet was evaluated on MALDI-MSI of mouse brain, DESI-MSI of renal tumor, and a synthetic data set with ground truth. It consistently outperformed state-of-the-art methods (e.g., t-SNE + k-means, CNNAE + region-growing, and GCN-based models), achieving higher accuracy and biological fidelity. On simulated data, MSInet achieved an Adjusted Rand Index of 0.89 and Normalized Mutual Information of 0.86, with ∼25.8% ARI improvement over baselines. It also precisely delineated complex anatomical subregions in the brain (Silhouette Coefficient = 0.78) and distinguished tumor, necrosis, and healthy regions in renal tissues, closely aligning with histological references. MSInet further demonstrated robustness to MSI noise. By integrating global and local contextual modeling in a self-supervised architecture, MSInet offers a powerful, scalable solution for accurate and biologically meaningful MSI segmentation, with broad potential for spatial omics and biomedical applications.

DOI: https://doi.org/10.1021/acs.analchem.5c04885
Molecules. 2025 Oct 14. pii: 4085. [Epub ahead of print]30(20):

An Innovative Use of the QuEChERs Method and LC-MS/MS Technique for Fast and Simple Determination of Quinolizidine Alkaloids in Leguminous Plants.

Ewa Rutkowska.

  Quinolizidine alkaloids, found particularly in leguminous plants (Fabaceae), are known for their role in plant protection, acting as toxic secondary metabolites against pests and pathogens. However, their toxicity also makes them anti-nutritional factors in food and feed. Therefore, it is necessary to monitor their presence. The aim of this study is to optimise two stages of the research procedure, i.e., (1) the conditions of LC-MS/MS instrumental analysis for the simultaneous determination of five alkaloids: angustifolin, hydroxylupanine, sparteine, and two geometric isomers of lupanine and isolupanine, and (2) the extraction and isolation stage of six different leguminous matrices: field beans, peas, lupins (narrow-leaved, white, yellow) and lentils. The modified and validated QuEChERS method based on LC-MS/MS shows acceptable recoveries (71-115%) with relative standard deviation <15%. A slight matrix effect (-20-14%) was observed. The uncertainty of the method <28%. The developed method shows significant progress in terms of sensitivity, achieving a detection limit as low as 0.01 mg/kg. This is a significant improvement over existing analytical methods and highlights the great potential of this method for detecting trace amounts. The innovative, sensitive, and selective method, offering simplicity and speed, was applied to the analysis of real leguminous samples.

Keywords:  LC-MS/MS; QuEChERS; analytical method; leguminous plants; optimisation; quinolizidine alkaloids

DOI:  https://doi.org/10.3390/molecules30204085
J Pharm Biomed Anal. 2025 Oct 05. pii: S0731-7085(25)00521-7. [Epub ahead of print]268 117180

Validated LC-MS/MS method for cisplatin quantification in plasma, whole blood, and cervical cancer tissue via diethyldithiocarbamate derivatization: Clinical application in cervical cancer.

Honghao Bai, Min-Gyo Seo, Seo-Yeon Choi, Vipada Khaowroongrueng, Yoon-Jee Chae, Ji-Young Jeon, Sun-Young Lee, Min-Gul Kim, Kwang-Hee Shin.

  The objective of this research was to establish an LC-MS/MS method with high sensitivity and selectivity for the quantification of cisplatin in human plasma, whole blood, and cervical cancer tissue samples. This approach employed diethyldithiocarbamate (DDTC) as a derivatizing agent for cisplatin, and analyte detection was conducted using the multiple reaction monitoring (MRM) mode. The method demonstrated lower limits of quantification (LLOQ) of 1 ng/mL for both plasma and tissue, while for whole blood, the LLOQ was determined to be 5 ng/mL. This method demonstrated remarkable linearity (R² >0.98), precision (coefficient of variation <15 %), accuracy (85 %-115 %), and minor matrix effects in all matrices. Stability assessments confirmed the robustness of the method under various conditions, including freeze-thaw cycles, short-term storage, and reinjection. Clinical samples from cervical cancer patients treated with intravenous 40 mg/m² cisplatin over 1 h revealed concentrations from below the LLOQ to 4250 ng/mL in plasma, 55-1673 ng/mL in whole blood, and 197-1613 ng/mL in tissue. The successful application of this method enables precise pharmacokinetic and tissue distribution studies of cisplatin, facilitating personalized dosing strategies to improve treatment outcomes for cervical cancer patients.

Keywords:  Cervical cancer; Cisplatin; LC-MS/MS; Plasma; Tissue; Whole blood

DOI:  https://doi.org/10.1016/j.jpba.2025.117180
Rapid Commun Mass Spectrom. 2026 Feb 15. 40(3): e10164

In Vitro Metabolite Profiling of 4,4-Diaminodiphenylmethane Using Human Liver S9 Fractions and LC-Orbitrap HRMS: In Silico Toxicity Studies of Its Metabolites.

Sridhar Chinthakindi, Radhika Kawathe, Leela Sairam Andhela.

   RATIONALE: 4,4-Diaminodiphenylmethane (MDA) is an aromatic amine classified as a Group 2B carcinogen. MDA is widely used as a key ingredient in industrial and consumer products such as epoxy resins, polyurethanes, and dyes. Given its widespread use and potential for occupational and environmental exposure, understanding its hepatic metabolism is crucial in elucidating the toxicological profiles of MDA in humans. However, a limited number of metabolites have been reported in the literature. To address this gap, we conducted a comprehensive metabolite profiling study to identify and characterize all possible in vitro metabolites of MDA.
METHODOLOGY: In vitro hepatic metabolism studies were conducted using pooled human liver S9 fractions to stimulate the metabolism of MDA in humans. An untargeted metabolite profiling approach was employed using an LC-Orbitrap-HRMS. Data processing and identification of metabolites were performed using the Compound Discoverer software. A targeted LC-MS/MS method was employed to find the relative abundances of metabolites at various incubation times. In silico toxicity prediction of MDA-derived metabolites was conducted using the ProTox 3.0 online tool.
RESULTS: A total of twelve MDA-derived metabolites were successfully identified, reflecting both Phase I (hydroxylation, desaturation, oxidative deamination) and Phase II (acetylation, sulfation, glucoside conjugation) biotransformation pathways. Among these, N-acetylated and desaturation intermediate-related metabolites were the most abundant. Based on these findings, an in silico toxicological report for the metabolites was proposed.
CONCLUSIONS: The Orbitrap LC-HRMS platform enabled comprehensive identification and characterization of in vitro hepatic metabolic profiles of MDA. The predicted toxicological profiles and the discovery of additional metabolite formation beyond the known acetylation pathway provided new insights that can enhance toxicological risk assessment of MDA.

Keywords:  LC‐Orbitrap‐HRMS; MDA; in silico toxicity; in‐vitro metabolism; liver S9 fractions

DOI:  https://doi.org/10.1002/rcm.10164
Front Endocrinol (Lausanne). 2025 ;16 1632694

Screening of biomarkers for early diagnosis of trauma-induced coagulopathy based on untargeted metabolomics.

Xu Xu, Qianhui Ouyang, Min Shao, Qi Yi, Sixiang Liu, Ying Huang, Jia Wang, Xingwen Zhang, Chaochao Tan.

   Objectives: Trauma-induced coagulopathy (TIC) is an acute coagulation disorder characterized by massive bleeding following trauma and is a leading cause of mortality. However, current clinical methods are inadequate for predicting TIC onset, and reliable biomarkers for early diagnosis are lacking. This study aimed to identify potential biomarkers with high sensitivity and specificity for TIC using an untargeted metabolomics approach.
Methods: We analyzed serum samples from 54 trauma patients (27 with TIC and 27 without TIC) and 27 healthy controls. All samples were collected within 24 hours post-trauma. Metabolomic profiling was conducted using liquid chromatography-tandem mass spectrometry (LC-MS).
Results: Metabolite profiles differed significantly between the TIC and non-TIC groups. Two metabolites, LysoPE(20:4(8Z,11Z,14Z,17Z)/0:0) (AUC = 0.933, 95% CI: 0.849-0.995) and LysoPE(0:0/18:2(9Z,12Z)) (AUC = 0.916, 95% CI: 0.818-0.914), were identified as potential biomarkers for distinguishing TIC. The diagnostic performance of these metabolites surpassed that of both conventional coagulation tests and admission assessment scores.
Conclusion: Two LysoPE metabolites were identified as promising biomarkers for the early detection of TIC.

Keywords:  liquid chromatography-tandem mass spectrometry (LC-MS); lysophosphatidylethanolamine (LysoPE); metabolomics; trauma; trauma-induced coagulopathy (TIC)

DOI:  https://doi.org/10.3389/fendo.2025.1632694
Anal Chem. 2025 Oct 25.

High-Frequency Microfluidic Fractionation for Compound-Resolved Bioactivity-Based Metabolomics.

Christian Geibel, Julian Schubert, Simon B Knoblauch, Albert Hernandez, Leonardo Boldt, Dana C Schneider, Stilianos Papadopoulos Lambidis, Giovanni Andrea Vitale, Jakub Fleischer, Manuela Haussmann, Harald Gross, Mingxun Wang, Heike Brötz-Oesterhelt, Daniel Petras.

Specialized metabolites represent a prolific source of potential drug candidates. However, the process from detecting bioactivity in a crude metabolite extract to unambiguously identifying the active agent is a tedious and expensive endeavor. Speeding up this procedure is crucial, as new drugs, such as antibiotics, are urgently needed. Furthermore, the systematic functional assessment of complex metabolome samples represents a key bottleneck in nontargeted metabolomics, which once solved, holds the potential to fundamentally advance our systematic understanding of biology. To tackle this central bioanalytical challenge, we developed a compound-resolved bioactivity-based metabolomics workflow that combines nontargeted liquid chromatography tandem mass spectrometry (LC-MS/MS), high frequency fractionation on microfluidic devices and subsequent readout with luminescent bioreporter strains. Central for this workflow is a custom high-speed (∼1 Hz frequency) fractionation device that spots the mobile phase onto a microfluidic paper-analytical device (μPAD) in parallel to MS/MS data acquisition. Subsequently, the μPAD can be overlaid with a bioreporter strain, which displays cellular stress by expressing luciferase. The luminescence signal can then be correlated to MS signals through their chromatographic profiles. We evaluated five different luciferase-expressing bioreporter strains which provide information about different antibacterial modes of action, and tested the workflow with different antibiotic standards and mixtures thereof, as well as crude extracts from the known antibiotic producer Saccharopolyspora erythraea. Our results demonstrated high sensitivity (up to 1 ng/spot, depending on compound and bioreporter) and the rapid identification of multiple antimicrobial compounds out of crude extracts, highlighting the practicality and high-throughput capability of this compound-resolved bioactivity-based metabolomics approach.

DOI: https://doi.org/10.1021/acs.analchem.5c04612
J Sep Sci. 2025 Nov;48(11): e70312

Cell Metabolomics Reveals the Hepatotoxic Mechanism of Copper in Normal Rat Liver Cells Using Reversed-Phase and Hydrophilic Interaction Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

Pangwei Liu, Shenao Zhang, Yuge Jiang, Huan Wu, Shijian Cao, Hongfei Wu, An Zhou.

  Copper (Cu) is an essential trace element for maintaining normal cellular functions; however, excessive Cu accumulation has been confirmed to induce hepatotoxicity, while the metabolic mechanisms underlying Cu-induced hepatotoxicity remain unclear. In this study, an innovative integrated separation strategy was established, combining hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF/MS), to systematically resolve metabolomic perturbations in CuCl2-exposed rat BRL-3A hepatocytes. Based on their complementary separation mechanisms-HILIC enables efficient retention and separation of polar metabolites via hydrophilic interactions, while RPLC separates nonpolar/weakly polar lipid molecules based on hydrophobic interactions-this analytical strategy significantly expanded the coverage of detectable metabolites and improved the reliability of metabolite identification through cross-validation between the two chromatographic platforms. The results showed that a total of 25 metabolites with significant changes were identified when BRL-3A cells were exposed to 50 µM CuCl2 (with a cell viability of 85%). These changes were mainly enriched in metabolic pathways such as glutathione metabolism (characterized by a significant decrease in the GSH/GSSG ratio, p < 0.01), arachidonic acid (AA) metabolism (a 42% reduction in AA, p < 0.05), and glycerophospholipid metabolism (a 1.8-fold increase in the levels of lysophospholipids [LysoPCs/LysoPEs], p < 0.05). These findings reveal that oxidative stress, membrane structure damage, and energy metabolism imbalance are the core mechanisms of Cu-induced hepatotoxicity. The integrated liquid chromatography-mass spectrometry (LC-MS) analytical framework established in this study not only provides a novel molecular perspective for elucidating the mechanisms of Cu-induced hepatotoxicity but also demonstrates the application potential of advanced complementary separation technologies in the risk assessment of environmental pollutants.

Keywords:  HILIC‐Q‐TOF/MS; RPLC‐Q‐TOF/MS; copper exposure; hepatotoxicity; metabolomics

DOI:  https://doi.org/10.1002/jssc.70312
Anal Chem. 2025 Oct 27.

AI-SNPS2: A Multi-Layered LC-MS/MS Platform Integrating Molecular Networking and Retention Time Prediction for Identifying Controlled and New Psychoactive Substances.

So Yeon Lee, Jihyun Lee, Jaewoo Song, Minhye Lee, Hang-Ji Ok, Eunyoung Han, Youngmin Hong, Han Bin Oh.

The emergence of unknown controlled substances poses a significant challenge in forensic and analytical sciences. While liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables identification of compounds through spectral database matching, it remains limited for synthesized analogues not present in existing libraries. To address this gap, we developed AI-SNPS2 (Artificial Intelligence Screener for Narcotic Drugs and New Psychoactive Substances 2), an enhanced version of our previously reported screening software. AI-SNPS2 is structured into five integrated layers: LC-MS Viewer, AI Classifier, Identifier, NetBuilder (a GNPS-inspired molecular networking module), and RT Predictor (a machine learning-based retention time prediction module). These layers allow structural analogue detection via spectral similarity and chromatographic plausibility filtering, thereby extending identification capabilities beyond conventional spectral search. The RT Predictor layer incorporates four regression models─artificial neural network (ANN), support vector regression (SVR), random forest (RF), and extreme gradient boosting (XGBoost)─trained on 42 molecular descriptors from 164 controlled substances. All models exhibited strong performance, with XGBoost achieving the highest accuracy (R2 = 0.964, MAE = 0.585). When applied with the RT calibration function, deviations were typically within a few minutes on a 110 min gradient, demonstrating the RT predictor's utility for candidate filtering. The software's utility was further evaluated by spiking JWH-019, JWH-015, and JWH-302 into two complex matrices; both were successfully identified through integration of molecular networking (MN) and hybrid similarity search (HSS) algorithm. Furthermore, evaluation using five additional compounds demonstrated that AI-SNPS2 is a highly promising tool for detecting compounds absent from existing databases.

DOI: https://doi.org/10.1021/acs.analchem.5c02830
Nat Commun. 2025 Oct 27. 16(1): 9479

Barcode-free hit discovery from massive libraries enabled by automated small molecule structure annotation.

Edith van der Nol, Nils Alexander Haupt, Qing Qing Gao, Benthe A M Smit, Martin Andre Hoffmann, Martin Engler-Lukajewski, Marcus Ludwig, Sean McKenna, J Miguel Mata, Olivier J M Béquignon, Gerard van Westen, Tiemen J Wendel, Sylvie M Noordermeer, Sebastian Böcker, Sebastian Pomplun.

Affinity-selection platforms are powerful tools in early drug discovery, but current technologies - most notably DNA-encoded libraries (DELs) - are limited by synthesis complexity and incompatibility with nucleic acid-binding targets. We present a barcode-free self-encoded library (SEL) platform that enables direct screening of over half a million small molecules in a single experiment. SELs combine tandem mass spectrometry with custom software for automated structure annotation, eliminating the need for external tags for the identification of screening hits. We develop efficient, high-diversity synthesis protocols for a broad range of chemical scaffolds and benchmark the platform in affinity selections against carbonic anhydrase IX, identifying multiple nanomolar binders. We further apply SELs to flap endonuclease 1 (FEN1) - a disease related DNA-processing enzyme inaccessible to DELs - and discover potent inhibitors. Taken together, screening barcode-free libraries of this scale all at once represents an important development, enables access to novel target classes, and promises substantial impact on both academic and industrial early drug discovery.

DOI: https://doi.org/10.1038/s41467-025-65282-1
J Pharm Biomed Anal. 2025 Oct 20. pii: S0731-7085(25)00548-5. [Epub ahead of print]268 117207

Analysis of growth hormone releasing hormone and its analogs in urine using nano liquid chromatography coupled with quadrupole/orbitrap mass spectrometry.

Ebru Uçaktürk, Emirhan Nemutlu.

  Growth hormone-releasing hormone (GHRH) and its synthetic analogs are considered performance-enhancing substances and are therefore prohibited by the World Anti-Doping Agency (WADA). The analysis of GHRH and its analogs in urine presents significant analytical challenges due to their inherent in vivo instability, rapid renal clearance, and low urinary concentrations. The present study aimed to develop a robust nano-LC quadrupole/orbitrap mass spectrometry (nano-LC-Q/Orbitrap MS) method for both screening and confirmation analyses of GHRH and its synthetic analogs (sermorelin/CJC-1293, tesamorelin, and CJC-1295) and the primary metabolite of sermorelin in urine, in accordance with WADA requirements. The sample preparation workflow was systematically investigated. Existing solid-phase extraction (SPE) protocols were compared, and two additional commercially available SPE cartridges were evaluated. Within the SPE step, the influence of various washing and elution solvent strengths on peptide recovery was also systematically examined. The effectiveness of different cleanup solvents during the ultrafiltration step was further assessed. Based on these evaluations, a refined approach was developed, incorporating an initial ultrafiltration step followed by SPE. The proposed method was fully validated according to WADA guidelines, assessing key parameters such as selectivity, reliability, limits of detection (LOD), carryover, limits of identification (LOI), robustness, autosampler stability, and matrix effects. The validation results confirmed the method's suitability and robustness for anti-doping testing. Achieved LODs (≤ 0.5 ng/mL) and LOIs (0.5-0.75 ng/mL) demonstrated sufficient sensitivity for effective detection and confirmation analysis of the target peptides in urine.

Keywords:  Doping control; GHRH analogs; Growth hormone-releasing hormone; Nano-LC quadrupole/orbitrap mass spectrometry; Urine

DOI:  https://doi.org/10.1016/j.jpba.2025.117207
J Am Soc Mass Spectrom. 2025 Oct 28.

Development and Modification of Ion Mobility Mass Spectrometry Instrumentation for Enhanced Analysis of Biological Molecules.

Jamie P Butalewicz, Jennifer S Brodbelt.

Ion mobility separates ion in the gas phase based on rotationally averaged cross section, a parameter often correlated with size, providing a versatile measurement strategy when integrated with mass spectrometry. The rapid growth in the field of ion mobility mass spectrometry has been catalyzed by numerous innovative advances in instrumentation that have improved resolution, sensitivity, and the ability to measure collision cross sections. These advances in ion mobility instrumentation and methods have been translated into many applications in the fields of metabolomics, lipidomics, proteomics, and structural biology. This Perspective focuses on developments in ion mobility instrumentation, spanning the impressive capabilities of commercial platforms to customized designs and modifications that establish new benchmarks at the frontiers of ion mobility mass spectrometry.

DOI: https://doi.org/10.1021/jasms.5c00222