bims-metlip 2026-02-01 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–02–01
35 papers selected by
Sofia Costa, Matterworks

Development and Validation of a Rapid LC-MS/MS Method for the Quantification of Contezolid in Human Plasma and Liver, and Its Application to a Pharmacokinetic Study in Liver Transplant Patients.
Validated LC-MS/MS Method for Vibegron Quantification in Human Plasma via a Selective Fragment Ion (m/z 135.1): Addressing Matrix Interference and Sample Instability.
Comprehensive evaluation of reversed-phase and hydrophilic interaction liquid chromatography - mass spectrometry for analysis of gangliosides.
Development and Validation of UHPLC-MS/MS Method for the Determination of SAX and 5-OH-SAX in Human Plasma and Its Application to a Pharmacokinetic Study.
Phospholipid Profiling Established by Structure-Rich Fragments for Molecular Species Level Shotgun Analysis.
Quantitative Determination and Stability of Trace Levels of Oxytocin in Human and Rhesus Macaque Urine by LC-MS/MS.
Comprehensive Curation and Harmonization of Small-Molecule MS/MS Libraries in Spectraverse.
Determination of 15 phthalates metabolites and 9 bisphenols in human serum by using high-performance liquid chromatography coupled with tandem mass spectrometry.
Two Comprehensive Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-MS/MS) Multi-Methods for Real-Time Therapeutic Drug Monitoring (TDM) of Five Novel Beta-Lactams and of Fosfomycin Administered by Continuous Infusion.
Opioid detection and quantification in plasma and oral fluid by LC-MS/MS.
STRIKER: a spectral metadata repairing tool for expanding the comprehensiveness of spectral libraries.
Integrated targeted and untargeted analysis of polar peptides in foods using hydrophilic interaction liquid chromatography-data-independent acquisition-mass spectrometry.
Evaluating Batch Correction Methods for Large-Scale Mass Spectrometry Imaging of Heterogeneous Tissues.
Gas Chromatography-Mass Spectrometry (GC-MS) in the Plant Metabolomics Toolbox: Sample Preparation and Instrumental Analysis.
mspms: an R package and GUI for multiplex substrate profiling by mass spectrometry.
Evaluation of portable atmospheric-pressure solids analysis probe-quadrupole mass spectrometry and UPLC-MS for the rapid screening of illicit substances in human urine.
Retrieval-Augmented Language Models Enable Scalable Chemical Source Classification in Metabolomics Workflows.
Development and validation of LC-MS/MS method for determining multi-residue pesticides in vegetables.
Efficient quantification of methazolamide in rat plasma using UHPLC-Q-Orbitrap MS for pharmacokinetic analysis.
Longitudinal, Intra-Individual Stability of Untargeted Plasma and Cerebrospinal Fluid Metabolites.
Comprehensive Analysis of Different Subtypes of Oxylipins to Determine a LC-MS/MS Approach in Clinical Research.
A Novel Compact Multi-Reflecting Time-of-Flight Mass Spectrometer.
Integrated HPLC-UV validation and LC-MS/MS optimization with molecular networking and chemometrics for advanced characterization of chlorogenic acids in Ilex guayusa.
Characterization of In Vitro Metabolic Profiles of Elacestrant in Rat and Human Liver Microsomes Using HPLC-MS/MS and HPLC-Q-Orbitrap-HRMS.
Sexual Hormones Determination in Biofluids by In-Vial Polycaprolactone Thin-Film Microextraction Coupled with HPLC-MS/MS.
Simultaneous Determination of N-(1,3-Dimethylbutyl)-N'-Phenyl-p-Phenylenediamine (6-PPD) and 6-PPD-Quinone (6-PPDQ) in Mouse Tissues After Intratracheal Administration Using a Validated LC-MS/MS Method.
Development and Application of an LC-MS/MS Method for Simultaneous Quantification of Azathioprine and Its Metabolites: Pharmacokinetic and Microbial Metabolism Study of a Colon-Targeted Nanoparticle.
Simultaneous quantification of cytidine, methylcytidine, and hydroxymethylcytidine by isotope-dilution LC-MS/MS with application to mouse liver samples.
Visualizing xenobiotics and metabolites in zebrafish by VUV-laser desorption post-ionization mass spectrometry imaging.
Mass Spectrometric Identification and Isomer Differentiation of Novichok-Related Ethanimidamides.
Outpacing Emerging Drug Threats: Validation of ToxBox Kits That Automate LC-MS/MS Analyses.
Relevance analysis of N-glycan variations in C2C12 cells and mouse serum under simulated microgravity using a quaternary phosphonium hydrazide labeling strategy-based mass spectrometry quantitation approach.
Quantitative Identification of Hydrolysis Products of Mustards by Novel Acetylation Method and Liquid Chromatography-High Resolution Mass Spectrometric Analysis.
Determination of R/S-enantiomers of methamphetamine and amphetamine in human hair with chiral stationary phase LC-MS/MS.
An optimized HPLC-MS/MS assay for uracil and dihydrouracil in plasma: Improving dihydropyrimidine dehydrogenase phenotyping for individualized fluoropyrimidine treatment.

Biomed Chromatogr. 2026 Mar;40(3): e70369

Development and Validation of a Rapid LC-MS/MS Method for the Quantification of Contezolid in Human Plasma and Liver, and Its Application to a Pharmacokinetic Study in Liver Transplant Patients.

Zhuoran Tian, Siyi Zhong, Huimin Wei, Anni Pan, Hengkai Zhu, Dan Zhu, Jianfang Lu, Jinghua Yu, Xingxing Diao, Li Zhuang.

  A rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantification of contezolid in human plasma and liver tissue. Sample preparation was performed by protein precipitation using linezolid as the internal standard. Chromatographic separation was achieved on an ACQUITY BEH C18 column (2.1 × 50 mm, 1.8 μm) with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), delivered at a flow rate of 0.5 mL/min. Quantification was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 409.1 → 269.1 for contezolid and m/z 338.1 → 296.2 for linezolid. The method showed excellent linearity over the ranges of 20-10,000 ng/mL in plasma and 100-50,000 ng/g in liver tissue. Full validation confirmed that the method met regulatory standards for accuracy, precision, sensitivity, selectivity, stability, matrix effects, carryover, and interference. The validated assay was successfully applied to a pharmacokinetic study of contezolid in liver transplant recipients. In conclusion, this robust, efficient, and cost-effective LC-MS/MS method provides a valuable tool for the determination of contezolid in clinical and pharmacokinetic research.

Keywords:  LC–MS/MS; contezolid; human plasma and human liver tissue; liver transplantation; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70369
Biomed Chromatogr. 2026 Mar;40(3): e70363

Validated LC-MS/MS Method for Vibegron Quantification in Human Plasma via a Selective Fragment Ion (m/z 135.1): Addressing Matrix Interference and Sample Instability.

Ashim Kumar Sen, Santosh Tawari, Ujashkumar Shah.

  A sensitive and robust LC-MS/MS method was developed and validated for the quantification of vibegron in human plasma, employing electrospray ionization in positive mode and multiple reaction monitoring (MRM). The precursor ion m/z 445.5 yielded two product ions: m/z 427.4 and m/z 135.1. Despite higher intensity, m/z 427.4 was excluded due to chromatographic interference, while m/z 135.1, previously unreported for vibegron, was selected for its superior selectivity and specificity. A stable isotope-labeled internal standard, vibegron 13C5 (m/z 450.3 → 139.1), was used to enhance precision and minimize matrix effects. Chromatographic separation was achieved on a Kromasil C18 column with a mobile phase of acetonitrile and 10-mM ammonium formate (pH 5.8), yielding a retention time of 1.33 min and total runtime of 3.00 min. Liquid-liquid extraction with ethyl acetate provided consistent recovery (87.7%), and sodium fluoride was added to inhibit enzymatic degradation. The method demonstrated excellent linearity (0.500-400.223 ng/mL), precision (0.58%-3.34%), and accuracy (98.26%-102.29%), using only 100 μL of plasma. Fully compliant with ICH M10 guidelines, the method is well suited for future pharmacokinetic, bioavailability, and bioequivalence studies.

Keywords:  LC‐MS/MS; sodium fluoride; vibegron; vibegron 13C4

DOI:  https://doi.org/10.1002/bmc.70363
J Chromatogr A. 2026 Jan 22. pii: S0021-9673(26)00061-0. [Epub ahead of print]1770 466731

Comprehensive evaluation of reversed-phase and hydrophilic interaction liquid chromatography - mass spectrometry for analysis of gangliosides.

Miroslav Lísa, Roman Hájek.

  Gangliosides are biologically significant molecules with essential physiological roles in the organism. They are increasingly studied as biomarkers for disease diagnosis and progression. Due to their extreme structural diversity and typically low abundance in biological samples, efficient and sensitive liquid chromatography-mass spectrometry (LC-MS) methods are required for their analysis. In this study, we present a comprehensive comparison of the performance of two widely used LC-MS approaches: reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC). Both methods were independently optimized following an identical protocol, with a focus on chromatographic performance and MS response. Notably, salt concentration had a pronounced impact on ganglioside retention, peak shape, isomer separation, and MS signal intensity in both chromatographic modes. In HILIC, adjusting the initial gradient composition and profile enabled effective separation of gangliosides from highly abundant matrix lipids. The RP method demonstrated superior ganglioside signal intensity and a 3.5-fold reduction in analysis time. Both approaches allowed the separation of gangliosides based on the number of sialic acid and saccharide residues. Additionally, the RP method enabled separation according to ceramide composition. We established rules for RP separation based on the number of carbon atoms in the ceramide moiety, which can be used to predict ganglioside retention times. Based on our findings, each method offers distinct advantages for specific analytical goals. The HILIC approach is better suited for untargeted ganglioside profiling using accurate m/z and interclass separation, while the RP method is ideal for sensitive, high-throughput targeted analysis in complex biological matrices.

Keywords:  Gangliosides; Hydrophilic interaction liquid chromatography; LC-MS; Lipidomics; Lipids; Retention behavior; Reversed-phase

DOI:  https://doi.org/10.1016/j.chroma.2026.466731
Biomed Chromatogr. 2026 Mar;40(3): e70372

Development and Validation of UHPLC-MS/MS Method for the Determination of SAX and 5-OH-SAX in Human Plasma and Its Application to a Pharmacokinetic Study.

Lanlan Qiu, Jinjin Zeng, Jingjing Chen, Di Liu.

  A highly sensitive and reliable ultrahigh performance liquid chromatography-tandem mass spectrometry method (UHPLC-MS/MS) has been developed for the quantification of saxagliptin (SAX) and its metabolite, 5-hydroxy saxagliptin (5-OH-SAX), in human plasma. Utilizing an ACE Excel 3 Super C18 column with electrospray ionization multiple reaction monitoring, this method achieves excellent linearity across the concentration ranges of 0.1-50 ng/mL for SAX and 0.2-100 ng/mL for 5-OH-SAX. The mean extraction recoveries are reported at 99.87% for SAX and 97.68% for 5-OH-SAX, with matrix effects maintained at 0.85 and 0.86, respectively. Both intraday and interday relative standard deviations are maintained below 15%. Furthermore, the developed method confirms appropriate short-term stability, satisfying the requirements for pharmaceutical analysis without residual effects. Significantly, this validated method applies to pharmacokinetic studies, providing crucial insights into the drug and metabolite behaviors during varying ingestion states, thus enhancing therapeutic efficacy and patient compliance through informed SAX administration.

Keywords:  5‐hydroxy saxagliptin; UHPLC‐MS/MS; human plasma; saxagliptin

DOI:  https://doi.org/10.1002/bmc.70372
Rapid Commun Mass Spectrom. 2026 Apr 30. 40(8): e70038

Phospholipid Profiling Established by Structure-Rich Fragments for Molecular Species Level Shotgun Analysis.

Rong Chen, Amber H Jannasch, Bruce R Cooper, Jonathan H Shannahan, Christina R Ferreira.

   RATIONALE: Accurate identification of phospholipid molecular species remains a major challenge in shotgun lipidomics because conventional tandem mass spectrometry typically resolves only one structural moiety at a time. This structural ambiguity limits confident lipid biomarker discovery and biological interpretation. Improving structural specificity without sacrificing analytical speed is therefore critical for lipidomics and disease-related studies.
METHODS: Electrospray ionization tandem mass spectrometry was performed using direct infusion on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. MRMs were designed based on structure-rich phospholipid fragments containing both the headgroup and one fatty acyl chain. Lipids were extracted from mouse liver and brain tissues and analysed without chromatographic separation, and normal-phase LC was used for lipid headgroup confirmation only.
RESULTS: Structure-rich MS/MS transitions enabled molecular species identification of both diacyl and ether phospholipids. 15 PUFA-containing phospholipids were identified as candidate biomarkers differentiating healthy and metabolic syndrome mouse livers, revealing opposing regulation among structurally similar species supported by complementary fragmentation and LC evidence. In mouse brains, three ether lipid biomarkers were discovered, including plasmalogens and plasmanyl lipids, with distinct disease-associated trends.
CONCLUSION: This study demonstrates that structure-rich MS/MS transitions substantially improve phospholipid structural specificity in shotgun lipidomics while maintaining high throughput. The method enables reliable identification of individual lipid species with minimal isomer interference and is readily compatible with existing workflows. This strategy offers a practical path toward more precise lipid biomarker discovery and mechanistic insight into metabolic disease.

Keywords:  mass spectrometry; metabolic syndrome; phospholipids; plasmalogens; shotgun lipidomics

DOI:  https://doi.org/10.1002/rcm.70038
Biomed Chromatogr. 2026 Mar;40(3): e70349

Quantitative Determination and Stability of Trace Levels of Oxytocin in Human and Rhesus Macaque Urine by LC-MS/MS.

Xing Zhang, Lori Fralick, Kelly Ethun.

  Oxytocin (OT) quantification in biological samples is vital for understanding its role in social behavior, emotion, and brain development in humans and animals. Urinary OT analysis has gained attention but is challenging due to variability in matrix effects, background interference, low concentrations, and stability concerns. This study developed an efficient LC-MS/MS method using mixed-mode solid-phase extraction (SPE) for sample preparation and serially coupled columns for separation. It achieved a lower limit of quantification (LLOQ) of 1 pg/mL, surpassing the sensitivity of previous methods. The method was fully validated per FDA guidance. The extraction recovery was > 80% in human urine. The precision was < 9%, and accuracy was between 101% and 109%. Partial validation for rhesus macaque urine showed accuracy ranged between 93% and 109%, with precision under 11%. Stability studies confirmed that OT in urine remains stable at room temperature for 4 h and at -80°C for 30 days. SPE-extracted samples were stable at 4°C for 24 h, and OT was unaffected by up to three freeze-thaw cycles. These results indicate that the LC-MS/MS method is accurate, consistent, and reliable for measuring OT in human and rhesus macaque urine and provide guidance for proper sample handling and storage.

Keywords:  LC–MS/MS; human urine; nonhuman primates; oxytocin; rhesus macaque urine

DOI:  https://doi.org/10.1002/bmc.70349
Anal Chem. 2026 Jan 25.

Comprehensive Curation and Harmonization of Small-Molecule MS/MS Libraries in Spectraverse.

Vishu Gupta, Hantao Qiang, Hsin-Hsiang Chung, Ehud Herbst, Michael A Skinnider.

Reference libraries of tandem mass spectra (MS/MS) are widely used for metabolite identification in untargeted metabolomics and to train machine-learning models for metabolite annotation. However, public spectral libraries are scattered across disparate databases and contain spectra that are of low resolution or quality, missing critical metadata, or which have chemically incoherent annotations. Addressing these issues requires extensive preprocessing and considerable expertise in mass spectrometry, which presents a significant barrier to investigators interested in developing their own machine-learning models. Here, we present Spectraverse, a comprehensive and extensively curated library of public MS/MS spectra from small molecules. We assembled reference spectra from both major repositories and previously overlooked resources and then developed a preprocessing pipeline to harmonize metadata, standardize chemical structures, and remove low-quality or redundant spectra. These efforts led us to identify previously undocumented pitfalls in existing public libraries that may have confounded prior comparisons of machine-learning models or conversely have caused valid MS/MS spectra to have been discarded from the training sets of these models. The resulting resource affords the most comprehensive coverage of chemical space of any machine-learning-ready library of MS/MS spectra to date while also expanding the coverage of adducts and ionization modes encountered in metabolomics experiments. We intend to maintain and expand Spectraverse in order to encompass the growing number of publicly available reference MS/MS spectra that can be expected to accumulate in the future.

DOI: https://doi.org/10.1021/acs.analchem.5c06256
J Chromatogr A. 2026 Jan 22. pii: S0021-9673(26)00058-0. [Epub ahead of print]1769 466728

Determination of 15 phthalates metabolites and 9 bisphenols in human serum by using high-performance liquid chromatography coupled with tandem mass spectrometry.

Yilin Liu, Juan Wen, Xu Zhang, Xiao Lin, Zhuangzhuang Feng, Dejun Bao, Xiaojian Hu, Ying Zhu.

  Endocrine disrupting chemicals (EDCs) are a class of emerging contaminants that have raised significant public health concerns. Human co-exposure to mixtures of specific EDCs, such as phthalate and bisphenols, and the associated health risks remain poorly understood. To support biomonitoring and epidemiological studies on these compounds, there is a critical need for sensitive and reliable methods for their quantification in human specimens. Herein, we developed and validated an analytical method based on ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of 15 phthalate metabolites and 9 bisphenols in human serum. A systematic comparison between liquid-liquid extraction (LLE) and high-efficiency matrix removal (HMR) clean-up approach revealed that HMR offered advantages in terms of extraction efficiency and matrix effect reduction, and was ultimately selected as the final clean-up method for this study. The developed method demonstrated satisfactory performance, with recoveries ranging from 81 % to 115 %, with the relative standard deviations (RSD) below 18 %. The calibration curves established using isotope-labeled internal standards exhibited good linearity, with correlation coefficients (R2) consistently above 0.997. The limits of detection (LODs) and limits of quantification (LOQs) were determined to be in the range of 0.01 ng/mL to 0.2 ng/mL and 0.04 ng/mL to 0.7 ng/mL, respectively. Both intra-day and inter-day precision were satisfactory, with RSDs values ranging from 4.2 % to 13 % and 6.9 % to 14 %, respectively. This sensitive analytical method was successfully applied to the determination of target analytes in human serum samples from 15 pregnant women, demonstrating its applicability for monitoring of these emerging contaminants in human serum.

Keywords:  Biomonitoring; Endocrine disrupting chemicals; Highly efficient matrix removal; Human serum; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2026.466728
Pharmaceutics. 2026 Jan 10. pii: 91. [Epub ahead of print]18(1):

Two Comprehensive Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-MS/MS) Multi-Methods for Real-Time Therapeutic Drug Monitoring (TDM) of Five Novel Beta-Lactams and of Fosfomycin Administered by Continuous Infusion.

Ilaria Trozzi, Beatrice Giorgi, Riccardo De Paola, Milo Gatti, Federico Pea.

  Background/Objectives: Therapeutic drug monitoring (TDM) of β-lactams (BL), BL/β-lactamase inhibitor (BLI) combinations (BL/BLIc), and of fosfomycin may play a key role in optimizing antimicrobial therapy and in preventing resistance development, especially when used by continuous infusion in critically ill or immunocompromised patients. Unfortunately, analytical methods for simultaneously quantifying multiple BL/BLIc in plasma are still lacking. Methods: The aim of this study was to develop and validate two rapid, sensitive, and accurate UPLC-qTOF-MS/MS methods for the simultaneous quantification of five novel β-lactam or β-lactam/β-lactamase inhibitor combinations (ceftolozane/tazobactam, ceftazidime/avibactam, meropenem/vaborbactam, cefiderocol, and ceftobiprole) along with fosfomycin. Methods: Human plasma samples were prepared by protein precipitation using methanol containing isotopically labeled internal standards. Chromatographic separation was achieved within 10-12 min using two Agilent Poroshell columns (EC-C18 and PFP) under positive and negative electrospray ionization modes. The method was validated according to the EMA guidelines by assessing selectivity, linearity, precision, accuracy, matrix effects, extraction recovery, and stability. Results: The methods exhibited excellent linearity (R2 ≥ 0.998) across the calibration ranges for all of the analytes (1.56-500 µg/mL), with limits of quantification ranging from 1.56 to 15.62 µg/mL. Intra- and inter-day precision and accuracy were always within ±15%. Extraction recovery always exceeded 92%, and the matrix effects were effectively corrected through isotopic internal standards. No carry-over or isobaric interferences were observed. All the analytes were stable for up to five days at 4 °C, but the BL and BL/BLIc stability was affected by multiple freeze-thaw cycles. Conclusions: These UPLC-qTOF-MS/MS multi-analyte methods enabled a simultaneous, reliable quantification in plasma of five novel beta-lactams and of fosfomycin. Robustness, high throughput, and sensitivity make these multi-methods feasible for real-time TDM, supporting personalized antimicrobial dosing and improved therapeutic outcomes in patients with severe or multidrug-resistant infections.

Keywords:  UPLC-qTOF-MS/MS; high resolution mass spectrometry; therapeutic drug monitoring (TDM); β-lactam antibiotics; β-lactamase inhibitors

DOI:  https://doi.org/10.3390/pharmaceutics18010091
Anal Bioanal Chem. 2026 Jan 28.

Opioid detection and quantification in plasma and oral fluid by LC-MS/MS.

Luana M Rosendo, Suzel Costa, Susana Simões, João M Franco, Noelia Serrano Gadea, Mónica Escorial, Francisco Javier Toboso Ortega, Segundo Jiménez-García, Ana M Peiró, Isabel Duque, Tiago Rosado, Mário Barroso, Eugenia Gallardo.

  The opioid crisis remains a significant public health concern, necessitating the development of sensitive and reliable analytical methods for drug detection. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of fentanyl, buprenorphine, oxycodone, morphine, tramadol, and tapentadol in plasma and oral fluid. The method was validated according to FDA guidelines, assessing selectivity, linearity, precision, accuracy, matrix effect, extraction efficiency, stability, carryover, and dilution integrity. The lower limits of quantification (LLOQs) were established at 0.1 ng/mL for fentanyl, 1.2 ng/mL for tramadol, and 0.6 ng/mL for the remaining opioids, demonstrating high sensitivity. The method exhibited excellent precision and accuracy, with coefficients of variation below 15% for intra-day, inter-day, and intermediate precision analyses. Extraction efficiencies exceeded 90% for most analytes, and matrix effects remained within acceptable limits. Real-world application to authentic plasma and oral fluid samples confirmed the method's robustness and reliability. Oral fluid concentrations were detectable across all target opioids, although plasma-oral fluid ratios showed some compound-dependent variability. These findings highlight the potential of oral fluid as a non-invasive complementary matrix to plasma for opioid monitoring, with relevant implications for forensic toxicology and clinical drug monitoring.

Keywords:  LC–MS/MS; Opioids; Oral fluid; Plasma

DOI:  https://doi.org/10.1007/s00216-026-06336-1
J Cheminform. 2026 Jan 27.

STRIKER: a spectral metadata repairing tool for expanding the comprehensiveness of spectral libraries.

Ahmed Karam, Asmaa Ramzy, Taghreed Khaled Abdelmoneim, Maha Mokhtar, Nada A Youssef, Aya Osama, Nabila Sabar, Sameh Magdeldin.

  The expansion of untargeted metabolomics has made publicly accessible spectral libraries indispensable for metabolite annotation and machine learning applications. Enhancing the quality and consistency of these libraries is crucial for improving the accuracy of metabolite identification and training machine learning models. However, public spectral libraries often suffer from variability in user submissions, unintentional errors, and a lack of standardization. Existing metadata cleaning and normalization tools typically exclude spectra with incorrect or unsupported metadata rather than attempting to correct them, resulting in the loss of valuable spectral data and associated metabolites details. This study introduces STRIKER (SpecTRal lIbrary maKER), a repair tool specifically designed to address adduct metadata deficiencies using a distance-based metric and a deep learning model. STRIKER leverages advanced similarity-based approaches to predict adducts in spectra lacking adduct metadata. It corrects adduct-related errors and standardizes adduct formatting using a deep learning model based on the multi-layer perceptron (MLP) algorithm. STRIKER achieved 95-99% correct adduct matching and 98% adduct correction accuracy. These corrections substantially reduce the number of missing or unusable spectra and metabolites, thereby enhancing the accuracy of metabolite identification and improving data quality for machine learning applications. The tool also facilitates a convenient construction of the Human Metabolome Database (HMDB) spectral library by integrating data files from the HMDB website. Furthermore, it enables users to extract customized sub libraries from larger libraries, supporting tailored analyses for specific research objectives with percised search space. STRIKER is an open-source, user-friendly Python graphical interface designed to be accessible to researchers with minimal bioinformatics expertise. Available at the following repository under an MIT license: https://striker-gui.sourceforge.io.Scientific contributionThe software is designed to preserve the maximum number of valid spectra in open mass spectral libraries, thereby supporting more comprehensive metabolite annotation in untargeted metabolomics. Its graphical user interface further facilitates the engagement of researchers without programming expertise, enabling them to enhance the quality and usability of spectral libraries.

Keywords:  Adduct correction; Adduct prediction; Library repairing; Mass spectrometry; Metadata; Python GUI; Spectral library; Untargeted metabolomics

DOI:  https://doi.org/10.1186/s13321-026-01150-4
Anal Bioanal Chem. 2026 Jan 28.

Integrated targeted and untargeted analysis of polar peptides in foods using hydrophilic interaction liquid chromatography-data-independent acquisition-mass spectrometry.

Boudewijn Hollebrands, Germaine Thong, Hans-Gerd Janssen.

  A hydrophilic interaction liquid chromatography-data-independent acquisition mass spectrometry (HILIC-DIA-MS) workflow was developed for simultaneous targeted semi-quantification of polar peptides and untargeted profiling of both peptides and other polar compounds in complex food matrices. The method uses a zwitterionic HILIC column optimized for separation of short polar peptides that are challenging to retain on reversed-phase columns. Many of these peptides contain charged amino acids and contribute to basic taste modalities such as umami and saltiness. Both targeted peptide analysis and comprehensive untargeted profiling were achieved by applying DIA-MS detection. This data acquisition mode was shown to be reproducible and sensitive while enabling retrospective data processing. High-resolution MS1 scans (60.000 FWHM), combined with fast MS2 scans and DIA mass windows of 15 m/z yielded highly repeatable and selective LC-MS profiles, allowing differentiation of structural isomers (e.g., alpha-glutamyl (umami) and gamma-glutamyl (kokumi)). The method was validated using taste-relevant dipeptides, demonstrating low detection limits (0.1-0.9 µM), good intra-day and inter-day precision, and high recovery (96%) in commercial soy sauce and yeast extract matrices. The workflow was further applied to the relative quantification of peptides and the untargeted profiling of characteristic molecular features in cheese, ham, and extracts from dried food ingredients. The integration of targeted and untargeted analyses demonstrates the suitability of HILIC-DIA-MS for comprehensive characterization of polar compounds in food systems.

Keywords:  Food peptides; HILIC-DIA-MS; Polar bioactive compounds; Retrospective analysis; Untargeted profiling

DOI:  https://doi.org/10.1007/s00216-026-06322-7
Anal Chem. 2026 Jan 26.

Evaluating Batch Correction Methods for Large-Scale Mass Spectrometry Imaging of Heterogeneous Tissues.

Martin Metodiev, Alex Dexter, Weiwei Zhou, Ariadna González-Fernández, Chelsea Nikula, Lucy M Johns, Evdoxia Karali, Emine Kazanc, Athanasios Tsalikis, Aurelien Tripp, Zoltan Takats, George Poulogiannis, Josephine Bunch.

In mass spectrometry imaging (MSI), the fluctuation in detected ion intensities, which is associated with "technical factors" and not the variability of molecular composition of the sample itself, may be referred to as "batch effects". These batch effects are a major barrier to the more widespread uptake and use of MSI for larger clinical and preclinical studies. In other fields, such as metabolomics and transcriptomics, batch correction methods have been introduced and commonly adopted. These methods aim to mitigate systematic biases introduced by differences in experimental conditions, instruments, or processing batches in high-dimensional data, such as omics or imaging data sets. Mass spectrometry imaging poses additional challenges compared to these fields such as the need to ensure that expected intensity fluctuations throughout a sample, associated with expected spatial variability, are maintained and the inability to randomly introduce quality control spectra. To date, there is no widely adopted approach to the batch correction of mass spectrometry imaging data. In this work, we consider both stabilization of intensity variability and the usefulness of correction methods for spatially resolved data. We present a pixel-by-pixel evaluation of batch correction for mass spectrometry imaging data.

DOI: https://doi.org/10.1021/acs.analchem.5c04371
Biomolecules. 2025 Dec 22. pii: 16. [Epub ahead of print]16(1):

Gas Chromatography-Mass Spectrometry (GC-MS) in the Plant Metabolomics Toolbox: Sample Preparation and Instrumental Analysis.

Nadezhda Frolova, Anastasia Orlova, Veronika Popova, Tatiana Bilova, Andrej Frolov.

  Metabolomics, which is typically referred to as the post-genomic methodology addressing low-molecular-weight metabolites, became a powerful tool in post-genomic research over the last two decades. Indeed, the state-of-the-art metabolomics relies on several well-established complementary platforms-nuclear magnetic resonance (NMR) spectroscopy, liquid and gas chromatography coupled on-line with mass spectrometry (LC- and GC-MS, respectively), and capillary electrophoresis-mass spectrometry (CE-MS). Among them, GC-MS represents one of the oldest and most well-established techniques currently employed in the metabolomics of volatile compounds and non-volatiles-polar low-molecular-weight metabolites, which can be efficiently converted in volatile form by comprehensive derivatization of polar functional groups. Currently, GC-MS is established as the principal analytical method for characterizing primary plant metabolism, although other methods also contribute significantly to determining the complete metabolite profile. Therefore, here, we address the role of GC-MS in plant metabolomics and its potential for the profiling of low-molecular-weight metabolites. Further, we comprehensively review the methods of sample preparation with special emphasis on extraction and derivatization approaches, which are currently employed to improve the method performance and its metabolome coverage.

Keywords:  GC-MS-based profiling; metabolomics platforms; plant metabolomics; primary metabolites

DOI:  https://doi.org/10.3390/biom16010016
BMC Bioinformatics. 2026 Jan 24.

mspms: an R package and GUI for multiplex substrate profiling by mass spectrometry.

Charlie Bayne, Brianna Hurysz, David J Gonzalez, Anthony O'Donoghue.

   BACKGROUND: Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) is a powerful method for determining the substrate specificity of proteolytic enzymes, which is essential for developing protease inhibitors, diagnostics, and protease-activated therapeutics. However, the complex datasets generated by MSP-MS pose significant analytical challenges and have limited accessibility for non-specialist users.
RESULTS: We developed mspms, a Bioconductor R package with an accompanying graphical interface, to streamline the analysis of MSP-MS data. Mspms standardizes workflows for data preparation, processing, statistical analysis, and visualization. The tool is designed for accessibility, serving advanced users through the R package and broader audiences through a web-based interface. We validated mspms using data from four well-characterized cathepsins (A-D), demonstrating that it reliably captures expected substrate specificities.
CONCLUSIONS: mspms is the first publicly available, comprehensive platform for MSP-MS data analysis downstream of peptide identification and quantification. It integrates preprocessing, normalization, statistical testing, and visualization into a single, transparent, and user-friendly framework, making it a valuable resource for the protease research community. The package is distributed via Bioconductor, and a graphical interface is available online for interactive use.

Keywords:  Bioconductor; Bioinformatics tools; Computational proteomics; Data visualization; Mass spectrometry data analysis; Multiplex substrate profiling; Protease specificity; R package

DOI:  https://doi.org/10.1186/s12859-026-06373-8
J Chromatogr A. 2026 Jan 20. pii: S0021-9673(26)00051-8. [Epub ahead of print]1770 466720

Evaluation of portable atmospheric-pressure solids analysis probe-quadrupole mass spectrometry and UPLC-MS for the rapid screening of illicit substances in human urine.

Doreen N B Chandra Siri, Si Ling Ng, Ngee Sing Chong, Philip J Marriott, Yong Foo Wong.

  Atmospheric-pressure solids analysis probe (ASAP) is an ambient pressure ionisation mass spectrometry technique that allows the direct analysis of liquids and solids, with or without sample preparation, producing rapid, efficient, and real-time results. In this study, portable ASAP-quadrupole mass spectrometry (ASAP-QMS) method in positive ion mode was demonstrated for the rapid analysis of 9 drugs of abuse (amphetamine, methamphetamine, oxycodone, methadone, morphine, 3,4-methylenedioxymethamphetamine, ketamine, 6-monoacetylmorphine, and cocaine), fortified in water and urine samples, with analysis time of ∼30 s. Initially, a direct analysis approach without sample preparation was applied to fortified water and urine samples. However, significant urine matrix effects were observed, leading to ion suppression and detector saturation. A simple solid-phase extraction (SPE) procedure was then applied to reduce matrix effects arising from the urine samples. The influence of cone voltages on the fragmentation and abundance of molecular ions for the illicit compounds was evaluated. The analytes exhibited limits of detection in the range of 0.3 - 2 µg mL-1, which were considerably higher than the UPLC-ESIMS (0.05 - 0.1 µg mL-1) method. Drug compounds identification was performed via MS spectrum matching against an in-house database. Comparative assessment with UPLC-ESI linear ion trap MS using United Nations Office on Drugs and Crime International Collaborative Exercises urine samples showed detection accuracies of 43% for ASAP-QMS and 100% for the UPLC-MS method. The lower accuracy of ASAP-QMS was attributed to the poor spectral library matching (similarity scores < 850) in complex urine matrices, particularly for low-concentration drug compounds. Nevertheless, portable ASAP-QMS (with SPE) achieved a higher greenness assessment score (0.54) compared to UPLC-MS (0.37), highlighting its potential as a rapid and environmentally friendly drug screening approach.

Keywords:  Ambient-pressure ionisation mass spectrometry; Atmospheric-pressure solids analysis probe; Cocaine; Illicit drugs; Urine

DOI:  https://doi.org/10.1016/j.chroma.2026.466720
Anal Chem. 2026 Jan 29.

Retrieval-Augmented Language Models Enable Scalable Chemical Source Classification in Metabolomics Workflows.

Prajit Rajkumar, Runbang Tang, Harshada Sapre, Jasmine Zemlin, Victoria Deleray, Jeong In Seo, Siddharth Mohan, Shipei Xing, Harsha Gouda, Yasin El Abiead, Shirley M Tsunoda, Haoqi Nina Zhao, Pieter C Dorrestein.

There is a growing need for scalable chemical classification to support the interpretation of exposomics and metabolomics data. While structural categorization has been largely automated, functional and exposure-based labeling of chemicals remains a manual and time-consuming process. Here, we present chemsource, a flexible framework that integrates large language models (LLMs) with retrieval-augmented generation (RAG) to automate chemical classification. chemsource retrieves descriptive text from Wikipedia or PubMed abstracts based on chemical names and prompts LLMs to assign user-defined categories based on the retrieved content. We demonstrate classification into five exposure categories: endogenous metabolites, food molecules, drugs, personal care products, industrial chemicals, and combinations of these possibilities. Benchmarking against manually curated labels for 4,953 compounds showed 75% overall agreement, with category-level recall exceeding 75% across all classes. Expert review indicated that most discrepancies could be attributed to prompt ambiguity and incomplete manual labels rather than model failure. To demonstrate the utility of chemsource in metabolomics workflow, we applied it to eight public untargeted metabolomics data sets, revealing distinct exposure patterns across human biospecimens, mouse tissues, environmental dust, and consumer product extracts. chemsource is customizable via prompt editing, enabling diverse classification tasks without requiring coding expertise. The tool is freely available as a Python package (https://pypi.org/project/chemsource/). Text retrieval is free; classification requires user-supplied LLM API access.

DOI: https://doi.org/10.1021/acs.analchem.5c05301
Food Chem. 2026 Jan 22. pii: S0308-8146(26)00278-5. [Epub ahead of print]505 148120

Development and validation of LC-MS/MS method for determining multi-residue pesticides in vegetables.

Renata de Oliveira Silva, Amanda Fonseca Lopes, Crisiana de Andrade Nobre, Ronaldo Ferreira do Nascimento.

  This work describes the development and validation of a method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) for the determination of 17 pesticides in cauliflower and broccoli samples. Ten cauliflower and broccoli samples were obtained from a commercial establishment in Fortaleza-CE, Brazil. The pesticides were extracted using a Quenchers method. The method validation involved selectivity, matrix effect, linearity and statistical tests, limit of detection (LOD) and limit of quantification (LOQ), precision and accuracy. The method showed a good sensitivity, matrix effects, data heteroscedasticity, significance of R and slope of the curves. The method LODm was of 2.0 μg kg-1. Precision reached ≤20% and accuracy ranged from 80 to 110% for medium concentration levels and from 70 to 120% for high concentration for most analytes, in accordance with pesticide residue guidelines. The acephate was detected in broccoli samples at level below the MRL established by EU (approximately 4.2 μg kg-1).

Keywords:  Broccoli; Cauliflower; Liquid chromatography; Mass spectrometry; Pesticides; QuEChERS

DOI:  https://doi.org/10.1016/j.foodchem.2026.148120
Anal Methods. 2026 Jan 26.

Efficient quantification of methazolamide in rat plasma using UHPLC-Q-Orbitrap MS for pharmacokinetic analysis.

Xing Wang, Qing-Lei Zou.

Methazolamide, a carbonic anhydrase inhibitor, is widely used in the treatment of acute angle-closure glaucoma, chronic open-angle glaucoma, secondary glaucoma, and for reducing intraocular pressure during ophthalmic surgeries. This study established and validated a highly selective and sensitive ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) analytical method for quantifying methazolamide in plasma. The separation of methazolamide and acetazolamide (internal standard) was conducted using a BEH Shield RP18 column. The mobile phase employed a gradient elution with acetonitrile (solvent A) and water containing 0.1% formic acid (solvent B). Data collection was executed in the positive ionization mode, employing comprehensive full MS/dd-MS2 on a Q-Orbitrap mass spectrometer. Methazolamide and acetazolamide (internal standard) were well resolved, with retention times of 2.63 min and 1.52 min, respectively. Methazolamide exhibited a robust linear correlation over a plasma concentration range of 10-5000 µg L-1, with a lower limit of quantification established at 10 µg L-1. The precision and accuracy were maintained below 8%, with methazolamide extraction recovery from plasma ranging between 86.16% and 96.06%. This research introduces an efficient method for the quantification of methazolamide. The validated method effectively assessed methazolamide pharmacokinetics in rat plasma following a 2 mg kg-1 oral dose.

DOI: https://doi.org/10.1039/d5ay00276a
Metabolites. 2025 Dec 30. pii: 35. [Epub ahead of print]16(1):

Longitudinal, Intra-Individual Stability of Untargeted Plasma and Cerebrospinal Fluid Metabolites.

Briana Rocha, Erin M Jonaitis, Alana Hamwi, Corinne D Engelman.

   BACKGROUND/OBJECTIVES: Longitudinal metabolomics analysis offers valuable insights into how metabolic pathways change according to age and health status. However, metabolite levels can fluctuate due to biological factors (e.g., age, diet, and health status) and technical factors (e.g., sample handling, storage times, and instrument performance), with some metabolites exhibiting greater sensitivity to these sources of variability than others. This study aimed to characterize the longitudinal and technical stability of untargeted plasma and cerebrospinal fluid (CSF) metabolites and to identify a subset that remains reliable over the extended time scales required for epidemiological research.
METHODS: Untargeted ultrahigh-performance liquid chromatography-mass spectrometry (LC-MS) metabolomic profiles were available from multiple visits in the Wisconsin Registry for Alzheimer's Prevention (WRAP) and Wisconsin Alzheimer's Disease Research Center (ADRC) studies. For this analysis, we constructed a subset of generally healthy participants with samples drawn at four time points (~2.5 years apart): two visits analyzed in 2017 and two visits analyzed in 2023, corresponding to two distinct analytical waves. We computed Rothery's intraclass correlation coefficients (ICCs) to quantify intra-wave and inter-wave stability, evaluated pooled quality-control (QC) variation, classified metabolite stability by established thresholds, and developed a composite score integrating longitudinal stability and susceptibility to technical variance.
RESULTS: Across all metabolites, median stability was classified as 'fair' (Rothery's ρ > 0.40 to ≤0.75) for both plasma and CSF. Although analytical batches were bridged using pooled QC samples, inter-wave stability was significantly lower than intra-wave stability, reflecting increased technical variability across waves. Using the composite score, we identified subsets of metabolites with 'excellent' stability and low susceptibility to batch effects in plasma and CSF. Stability patterns varied across biochemical super pathways.
CONCLUSIONS: This work highlights metabolites suitable for long-term epidemiological studies and informs experimental design and analytical strategies for combining data across cohorts and analytical batches.

Keywords:  batch effects; biomarker stability; cerebrospinal fluid; intraclass correlation coefficient; longitudinal metabolomics; metabolite stability; plasma

DOI:  https://doi.org/10.3390/metabo16010035
Metabolites. 2025 Dec 22. pii: 4. [Epub ahead of print]16(1):

Comprehensive Analysis of Different Subtypes of Oxylipins to Determine a LC-MS/MS Approach in Clinical Research.

Yurou Zhao, Zhengyu Fang, Zeyu Li, Yizhe Liu, Yang Bai, Xiaoqing Wang, Hongjun Yang, Na Guo.

   BACKGROUND/OBJECTIVES: Different oxylipin subtypes have unique biological properties, requiring effective analytical protocols. However, establishing a complete pathway detection protocol for comprehensive oxylipin analysis is challenging. This study aimed to evaluate the adaptability and specificity of oxylipin subtypes under different extraction schemes and to develop a robust analytical platform for clinical biomarker investigation.
METHODS: We revealed the adaptability and specificity of oxylipin subtypes based on different single-step extraction schemes. A high-throughput quantitative automated solid-phase extraction coupled with a liquid chromatography-tandem mass spectrometry (aSPE-LC-MS/MS) analytical platform was established for a broad panel of complex oxylipins. The method was applied to serum samples of patients with coronary heart disease (CHD).
RESULTS: Our results verified that oxo-oxylipins, resolvin, and eicosanoids showed the best extraction efficiency under SPE protocol. Most hydroxy-oxylipins, dihydroxy-oxylipins, and HOTrEs are suitable for methanol protocol, HDHA for acetonitrile protocol, and epoxy-oxylipins for the methyl tert-butyl ether protocol, while medium-chain HETE is suitable for ethyl acetate protocol. Importantly, a novel sensitive fast method with wide coverage by the aSPE-LC-MS/MS analytical platform with satisfying sensitivity, accuracy and precision, extraction efficiency, low matrix effect, and linear calibration curves was obtained. Furthermore, we have successfully applied this method and found that 5-HETE, 11-HETE, and 15-HETE can serve as integrated biomarkers for patients with CHD, with high diagnostic performance.
CONCLUSIONS: The study provides the best protocol for the clinically targeted detection of oxylipins and provides an important means for studying biomarkers of diseases.

Keywords:  clinical protocols; extraction protocol; extraction recovery rate; matrix effect; oxylipin

DOI:  https://doi.org/10.3390/metabo16010004
J Am Soc Mass Spectrom. 2026 Jan 26.

A Novel Compact Multi-Reflecting Time-of-Flight Mass Spectrometer.

Anatoly N Verenchikov, Jason Wildgoose, Sergey N Kirillov, Aleksey V Vorobyev, Vasily V Makarov, Lee A Gethings, Robert P Tonge, Matthew E Daly, William J Johnson, James I Langridge.

  Mass spectrometry is an indispensable tool for the rapid and in-depth analysis of complex mixtures across diverse biologically important fields including metabolomics, lipidomics, and proteomics. These applications demand high speed instruments with subppm mass measurement accuracy over a wide dynamic range of sample concentrations. Here, we introduce an liquid chromatography-mass spectrometry/MS (LC-MS/MS) quadrupole time-of-flight mass spectrometer featuring a novel collision cell, a high dynamic range detector, and a compact multireflecting orthogonal time-of-flight analyzer. This innovative instrument achieves high analytical performance, acquiring full mass range spectra at 100,000 Full Width Half Maximum (FWHM) resolution up to 100 spectra/s acquisition speed. The instrument achieves excellent linearity within a dynamic range of 105, with a correlation coefficient R2 = 0.984. The speed, resolution and dynamic range are in excellent balance as demonstrated by the analysis of isotopically labeled lipids in human blood plasma.

Keywords:  high resolution; lipidomics; multireflecting time-of-flight

DOI:  https://doi.org/10.1021/jasms.5c00321
J Chromatogr A. 2026 Jan 22. pii: S0021-9673(26)00062-2. [Epub ahead of print]1769 466732

Integrated HPLC-UV validation and LC-MS/MS optimization with molecular networking and chemometrics for advanced characterization of chlorogenic acids in Ilex guayusa.

Thomas Garzón, Jefferson V Pastuña-Fasso, Cristian Quiroz-Moreno, Melanie Ochoa-Ocampo, Evencio J Medina-Villamizar, Nina Espinosa de Los Monteros-Silva, Karel Diéguez-Santana, Jessica L Cooperstone, Zulay Niño-Ruíz, José R Almeida, Noroska G S Mogollón.

  Chlorogenic acids (caffeoylquinic acid isomers, CQAs) are major phenolic constituents of Ilex guayusa, but their comprehensive profiling in complex plant matrices is hindered by co-elution, overlapping UV spectra, and isomeric similarity in MS/MS. Rather than aiming to fully resolve isomer-specific quantification by MS, here we present an integrated workflow that couples validated HPLC-UV quantification of the major CQA (5-CQA) with an optimized UPLC-MS/MS strategy designed to improve MS1 peak integrity and expand MS/MS coverage for higher-confidence structural annotation. The HPLC-UV method showed excellent performance for targeted quantification of 5-CQA, including strong linearity (r² = 0.998), selectivity, sensitivity (LOQ = 0.25 mg/L), precision, and recovery. For LC-MS/MS, FastDDA acquisition (top-5 vs. top-15 precursors) revealed the expected trade-off between fragmentation depth and MS1 peak quality; however, post-acquisition raw-data merging restored MS1 fidelity and increased the number of detected features by 43%, enabling high-confidence annotation rather than quantitative discrimination of 16 metabolites and the propagation of oxidized CQA-related derivatives using feature-based molecular networking. Multivariate analyses (PCA, volcano plots, HCA) indicated that geographic location exerted the strongest influence on the metabolite composition, followed by sunlight exposure and plant age. Overall, the proposed workflow provides a practical framework that integrates robust chromatographic quantification with MS acquisition and data-processing optimization, thereby enhancing structural characterization and biological interpretation, rather than complete isomer-resolved quantification, of chlorogenic-acid-related chemistry across complex plant-derived and natural product matrices.

Keywords:  Ilex guayusa; Metabolomic fingerprinting; Metabolomics; Validation of method

DOI:  https://doi.org/10.1016/j.chroma.2026.466732
Biomed Chromatogr. 2026 Mar;40(3): e70374

Characterization of In Vitro Metabolic Profiles of Elacestrant in Rat and Human Liver Microsomes Using HPLC-MS/MS and HPLC-Q-Orbitrap-HRMS.

Min Hu, Fang Wang, Wenxia Liu, Shangxiu Chen, Yonggang Chen.

  Elacestrant is an orally administered selective estrogen receptor degrader designed for the treatment of ER-positive, HER2-negative breast cancer. In this study, a simple and sensitive HPLC-MS/MS method was developed and validated for the quantification of elacestrant in liver microsomes. Chromatographic separation was achieved on a Waters ACQUITY BEH C18 column using a gradient of 0.1% formic acid in water and acetonitrile. Elacestrant and the internal standard (elacestrant-d6) were detected in multiple reaction monitoring mode via the transitions of m/z 459.2 → 298.3 and m/z 465.3 → 304.4, respectively. The method exhibited excellent linearity over the range of 1.0-2000 nM (r > 0.995). Elacestrant was rapidly metabolized, showing half-lives of 22.45 ± 0.66 min in rat liver microsomes and 43.36 ± 2.48 min in human liver microsomes. Using HPLC-Q-Orbitrap-HRMS, seven metabolites were identified, with M5 (N-deethylation) being the most abundant. Key metabolic pathways involved O-demethylation, N-deethylation, N-dealkylation, and oxidative deamination, primarily mediated by cytochrome P450 3A4. This study establishes the first HPLC-MS/MS and HPLC-Q-Orbitrap-HRMS-based analytical strategy for in vitro metabolic profiling of elacestrant, supporting its future application in clinical pharmacokinetic and metabolism investigations.

Keywords:  cytochrome P450 3A4; elacestrant; metabolic stability; metabolite identification; selective estrogen receptor degrader

DOI:  https://doi.org/10.1002/bmc.70374
Molecules. 2026 Jan 12. pii: 255. [Epub ahead of print]31(2):

Sexual Hormones Determination in Biofluids by In-Vial Polycaprolactone Thin-Film Microextraction Coupled with HPLC-MS/MS.

Francesca Merlo, Silvia Anselmi, Andrea Speltini, Clàudia Fontàs, Enriqueta Anticó, Antonella Profumo.

  The in-vial microextraction technique is emerging as an alternative sample treatment, as it integrates sorbent preparation, adsorption, and desorption of analytes in a single device before instrumental analysis. In this work, the applicability of polycaprolactone polymeric film, recently used for the in-vial microextraction of sex hormones from environmental waters, is studied in a low-capacity format for unconjugated sex hormones determination in biological samples by HPLC-MS/MS. Its performance was evaluated in urine and serum, achieving extraction in a short time (10 and 30 min, in turn) and satisfactory elution with ethanol, with recovery in the range of 65-111% in urine, 55-122% in bovine serum albumin (BSA) solution, and 66-121% in fetal bovine serum (FBS). In the case of protein matrices, a dilution to 20 g L-1 protein content and washing step (3 × 1 mL ultrapure water) afore the elution are required to achieve clean extract, as verified by a Bradford assay. Matrix-matched calibration was used for quantification, obtaining correlation coefficients greater than 0.9929; limits of detection and quantification were in the range of 0.01-0.65 and 0.03-1.96 ng mL-1 in urine, 0.02-0.8 and 0.05-2.5 ng mL-1 in BSA, and 0.02-1.0 and 0.06-3.0 g mL-1 in FBS, respectively. The in-vial polycaprolactone film proved to be reusable for several cycles (up to ten), and the greenness assessment revealed a good adhesion to green sample preparation principles. All these achievements further strengthen its feasibility for efficient extraction/clean-up of trace sex hormones in complex biological samples.

Keywords:  bioanalysis; estrogens; green sample preparation; in-vial microextraction; polymeric film; progestins; serum; urine

DOI:  https://doi.org/10.3390/molecules31020255
Biomed Chromatogr. 2026 Mar;40(3): e70367

Simultaneous Determination of N-(1,3-Dimethylbutyl)-N'-Phenyl-p-Phenylenediamine (6-PPD) and 6-PPD-Quinone (6-PPDQ) in Mouse Tissues After Intratracheal Administration Using a Validated LC-MS/MS Method.

Moongi Ji, Byeongchan Choi, In-Hyeon Kim, Je-Hein Kim, Se-Woong Park, Moonjung Hyun, Sung-Hwan Kim, Man-Jeong Paik.

  N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD), a tire rubber antioxidant, and its oxidation product 6-PPD-quinone (6-PPDQ) have raised environmental and health concerns due to their widespread occurrence and toxicity. This study aimed to develop and validate a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 6-PPD and 6-PPDQ in murine tissues. The method showed excellent linearity (r ≥ 0.9977), low detection limits (0.056-0.967 ng/mL), and high accuracy and precision across multiple tissue matrices, enabling reliable quantification at trace levels. Following repeated intratracheal administration in mice, both analytes were detected in the liver, lungs, and kidneys, but not in the spleen. Notably, 6-PPDQ exhibited marked dose-dependent accumulation in the lungs, whereas 6-PPD remained at low levels, suggesting strong pulmonary retention. In contrast, trace levels of 6-PPD and a dose-dependent increase in 6-PPDQ were found in the kidneys, indicating renal elimination as a clearance route. These results reveal selective organ distribution, with the lung as the primary deposition site, and highlight 6-PPDQ as a potential multi-organ toxicant. The validated LC-MS/MS method provides a valuable analytical platform for future toxicokinetic, biomonitoring, and mechanistic studies of tire-derived contaminants.

Keywords:  6‐PPD‐quinone (6‐PPDQ); LC–MS/MS; N‐(1,3‐Dimethylbutyl)‐N′‐phenyl‐p‐phenylenediamine (6‐PPD); mouse tissues

DOI:  https://doi.org/10.1002/bmc.70367
Pharmaceuticals (Basel). 2025 Dec 26. pii: 58. [Epub ahead of print]19(1):

Development and Application of an LC-MS/MS Method for Simultaneous Quantification of Azathioprine and Its Metabolites: Pharmacokinetic and Microbial Metabolism Study of a Colon-Targeted Nanoparticle.

Jingjing Zhang, Jiaqi Han, Ning Sun, Yuhan Zhu, Dong Mei, Libo Zhao.

  Background/Objectives: Given the clinical limitations of azathioprine (AZA) in treating inflammatory bowel disease, this study developed an AZA-loaded microbiota-modulating and colon-targeted nanoparticle constructed from pectin, Zein, and Eudragit®S100 (APZE), which was hypothesized to enhance efficacy while reducing toxicity. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to simultaneously quantify AZA and its metabolites, enabling the investigation of the pharmacokinetic and microbial metabolism differences between APZE and AZA suspension (AZAS). Methods: APZE was characterized, and an LC-MS/MS method was developed for quantifying AZA and its metabolites in multiple matrices. Given the potential of APZE for colon targeting and modulation of the microbiota, which may affect drug absorption, distribution, and microbiota-mediated metabolism, we determined analyte concentrations in rat plasma, tissues, and microbial cultures at different time points following administration of APZE or AZAS. Results: AZA, 6-mercaptopurine (6-MP), 6-methylmercaptopurine (6-MMP), and 6-thioguanine (6-TG) were quantified in positive ion mode, and 6-thiouric acid (6-TU) in negative ion mode. The assay demonstrated excellent accuracy, precision, and stability over the concentration range of 5-1000 ng/mL. Orally administered APZE exhibited higher bioavailability, improved intestinal absorption, and reduced formation of the inactive metabolite 6-TU compared to AZAS. In microbial cultures, AZA was metabolized primarily to 6-MP, and APZE underwent more extensive metabolism to 6-MP than AZAS. Conclusions: This method provides accurate and precise quantification of physiologically relevant concentrations of AZA and its metabolites (6-MP, 6-MMP, 6-TG, and 6-TU), offering a bioanalytical tool for the pharmacokinetic and gut microbiota metabolism studies of AZA formulations. These findings suggest that APZE is a promising drug delivery formulation.

Keywords:  LC-MS/MS; azathioprine; microbial metabolism; nanoparticles; pharmacokinetics

DOI:  https://doi.org/10.3390/ph19010058
J Chromatogr A. 2026 Jan 21. pii: S0021-9673(26)00052-X. [Epub ahead of print]1769 466721

Simultaneous quantification of cytidine, methylcytidine, and hydroxymethylcytidine by isotope-dilution LC-MS/MS with application to mouse liver samples.

Pooja Mishra, Jing Ma, Huan Xie, Fang Li, Robert Y L Tsai, Dong Liang.

  DNA methylation and hydroxymethylation are important epigenetic modifications that play key roles in cancer development and aging processes by regulating gene expression and genome stability. Traditionally, bisulfite conversion-based or antibody-based enzyme-linked immunosorbent assays are used to find DNA methylation. These tests are non specific, tedious, and not able to differentiate the difference between methylation and hydroxymethylation. To address these issues, we developed a sensitive, reproducible, and specific LC-MS/MS method for simultaneous quantification of two major DNA methylation products, 5-methyl-2'-deoxycytidine (5-mdC) and 5-hydroxymethyl-2'-deoxycytidine (5-hmdC), as well as 2'-deoxycytidine (2-dC), using corresponding stable isotope-labeled internal standards: 5-methyl-2'-deoxycytidine-d₃, 5-(hydroxymethyl)-2'-deoxycytidine-d₃, and 2'-deoxycytidine-¹³C,¹⁵N₂. We purified DNA samples from mouse liver tissue, broke them down with enzymes, filtered them, added internal standards, and then run them through a SCIEX 6500+ Triple Quad LC-MS/MS system with an Atlantis T3 C18 column under a binary gradient. The method showed great chromatographic separation and specificity, with MRM transitions of m/z 228.154 to 112.1 for 2-dC, 242.143 to 126.2 for 5-mdC, and 258.135 to 142.1 for 5-hmdC.Peak area ratio of analyte to internal standard exhibited linearity across calibration ranges of 5-5000 ng /mL for 2-dC, 0.5-500 ng/ mL for 5-mdC, and 0.05-10 ng/mL for 5-hmdC (R² > 0.999), using 2 µL injection and a total runtime of 9 min. The 5-hmdC level in female mouse liver significantly increased with aging from two to sixteen months old (0.0958 % to 0.1984 %; P<0.001), whereas 5-mdC remained unchanged (3.47 % to 3.56 %; n.s.). These data confirm the accurate and reproducible quantification of DNA methylation and hydroxymethylation in tissue samples using the developed LC-MS/MS assay and indicate a broad application to cell culture and clinical biomarker studies.

Keywords:  5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 2′-deoxycytidine; Aging; Bioanalytical validation; DNA hydroxymethylation; DNA methylation; LC–MS/MS; Liver tissue

DOI:  https://doi.org/10.1016/j.chroma.2026.466721
Anal Bioanal Chem. 2026 Jan 26.

Visualizing xenobiotics and metabolites in zebrafish by VUV-laser desorption post-ionization mass spectrometry imaging.

Anan Li, Shijie Lu, Xueqing Shang, He Zhang, Min Xie, Qiao Lu, Yongjun Hu.

  Mass spectrometry imaging (MSI) enables spatial mapping of chemical distributions across complex surfaces, with broad applications spanning materials analysis, life sciences, and clinical diagnostics. However, conventional ionization sources often suffer from limited ionization efficiency, creating a trade-off between detection sensitivity and spatial resolution. Post-ionization technology addresses this constraint by incorporating a secondary ionization step, significantly enhancing ionization yield and establishing itself as a leading approach for advancing mass spectrometry techniques. In this study, we have independently developed a vacuum ultraviolet-laser desorption post-ionization mass spectrometry (VUV-LDPI-MS) system, which significantly enhances detection sensitivity through the implementation of post-ionization technology. By integrating low-photon-energy long-wavelength laser desorption with the soft-ionizing capability of VUV light, this technique enhances the detection of chemical species within biological tissues-including both exogenous drugs and endogenous metabolites. The method achieved a theoretical detection limit of 8.3 pg/spot for methylene blue (MB), while markedly reducing substrate effects and sample background interference. Imaging results revealed that endogenous compounds closely aligned with zebrafish tissue anatomy, whereas the spatial distribution of exogenous MB strongly correlated with optical images. Successful application to zebrafish tissue sections enabled clear visualization of both MB and intrinsic metabolites. VUV-LDPI-MSI offers superior ionization efficiency with minimal sample preparation, presenting a robust alternative to conventional LDI-MS approaches and holding considerable promise for future technological development.

Keywords:  Laser desorption post-ionization; Mass spectrometry imaging; Vacuum ultraviolet light; Zebrafish

DOI:  https://doi.org/10.1007/s00216-026-06344-1
J Mass Spectrom. 2026 Feb;61(2): e70028

Mass Spectrometric Identification and Isomer Differentiation of Novichok-Related Ethanimidamides.

Havva Bekiroğlu Ataş.

  Chemical warfare agent (CWA) analysis faces a significant challenge when degradation or precursor compounds lack reference spectra in databases. This study focuses on three structural isomers, N-methyl-N-propylethanimidamide, N-methyl-N-isopropylethanimidamide, and N,N-diethylethanimidamide, which are relevant hydrolysis by-products/precursors of Novichok nerve agents (Schedule 1.A.13/1.A.14) yet are absent from standard spectral libraries. All three isomers were synthesized and then comprehensively characterized by gas chromatography-electron ionization mass spectrometry (GC-EI-MS), gas chromatography-chemical ionization mass spectrometry (GC-CI-MS), and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Distinctive fragmentation signatures were identified for each isomer. Trimethylsilyl derivatization of the ethanimidamides enhanced GC performance and revealed additional diagnostic ions. LC-HRMS results confirmed the fragment formulas and allowed unambiguous differentiation based on key ion intensity ratios. This work provides the first systematic mass spectral data for these Novichok-related ethanimidamides. The value of these reference spectra was demonstrated in the 57th OPCW Proficiency Test (PT-57): an unknown spiking chemical was successfully identified as N-methyl-N-propylethanimidamide by comparison to the generated spectra. The study thus delivers new analytical capabilities for CWA forensics-enabling confident identification of challenging isomeric degradation products through a combination of tailored derivatization, multimode mass spectrometry, and expert interpretation of fragmentation pathways.

Keywords:  Novichoks; chemical warfare agents; ethanimidamides; isomer differentiation; mass spectral interpretation

DOI:  https://doi.org/10.1002/jms.70028
Chem Res Toxicol. 2026 Jan 30.

Outpacing Emerging Drug Threats: Validation of ToxBox Kits That Automate LC-MS/MS Analyses.

Marina Avram, Isabel L Ritter, Omran Muslin, Snehal Parikh, Yasin M Ahmed, Marie I Gosselin, William E Fantegrossi, Laura P James, Gregory W Endres, Joseph M Reed, Jeffery H Moran.

The Centers for Disease Control and Prevention (CDC) recently began addressing critical informational gaps associated with the emergence of novel psychoactive substances (NPSs) through the formation of the Overdose Data to Action (OD2A) biosurveillance program. This program uses public health laboratories (PHLs) to report drug trends in nonfatal and fatal overdose cases, but meeting the OD2A program's goal of reporting use trends in near real-time is a challenge for resource-limited PHLs. New technology that automates NPS testing will help PHLs meet this goal and sustain OD2A testing. This study uses commercially available test kits capable of automating clinical and forensic testing platforms to validate a single liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical procedure for opioids, barbiturates, benzodiazepines, cannabinoids, stimulants, and several other drug classes. Kits incorporated Suspended-State and Just-Go technologies to stabilize NIST-traceable standards and enzymatic buffers at precise concentrations in a format that does not require sample cleanup or dilution prior to LC-MS/MS analysis. OD2A test kits were activated at 37 °C prior to the addition of 10 μL sample aliquots, and hydrolysis of conjugated urinary metabolites was complete after a 90 min incubation inside the autosampler held at 37 °C. Samples were injected directly on the column for LC-MS/MS analysis and method validation. Accuracy, precision, measurement uncertainty, calibration models, reportable range, sensitivity, specificity, carryover, interference, ion suppression/enhancement, and analyte stability met the testing requirements established for toxicology laboratories. Suitability studies using fortified human urine demonstrated that the automated LC-MS/MS method validated for OD2A biosurveillance produces highly accurate and precise results. The LC-MS/MS method was also successfully transferred and validated in an independent state public health laboratory to demonstrate how this new technology can be used to support a national biosurveillance program. This new approach will enable PHLs to provide actionable data in a timely manner.

DOI: https://doi.org/10.1021/acs.chemrestox.5c00389
Anal Bioanal Chem. 2026 Jan 28.

Relevance analysis of N-glycan variations in C2C12 cells and mouse serum under simulated microgravity using a quaternary phosphonium hydrazide labeling strategy-based mass spectrometry quantitation approach.

Qian-Ru Ma, Jie-Cheng Lu, Yan-Qing Li, Hong-Jie Lan, Yan Liu, Yu-Fen Zhao.

  Mass spectrometry combined with stable isotope labeling is a powerful technique for detecting disease-related changes in glycosylation patterns and identifying potential biomarkers. However, stable isotope labeling reagents that simultaneously offer high sensitivity, low cost, and stable sialic acid modifications remain scarce. In this study, we developed a convenient and cost-effective microwave-assisted method for synthesizing a stable isotopic quaternary phosphonium hydrazide labeling reagent pair, 14N/15N-P4HZD, for the quantitation difference analysis of N-glycans using HPLC-ESI-HRMS with high sensitivity and convenience. This strategy features high labeling efficiency, excellent reproducibility, and strong linearity (R2 = 0.9984) within a dynamic range spanning two orders of magnitude. The reagent pair is compatible with multiple ion source mass spectrometers and front-end chromatographic separation technologies. In particular, it enhances the ionization efficiency of sialylated N-glycans and facilitates their detection. The relative quantification method has been effectively applied to analyze the variations in N-glycomic profiles from two muscular atrophy models induced by simulated microgravity, specifically the C2C12 cell and hindlimb unloading mouse serum. We discover that these variations display characteristic relevance in both models. N-Glycans Man3GlcNAc3Fuc1 and Man3GlcNAc4Gal1Fuc1Sia1 exhibit their potential as biomarkers for the early diagnosis of muscular atrophy. The mass spectrometry method based on the 14N/15N-P4HZD reagent pair offers a convenient and feasible strategy for the difference analysis of N-glycomics, demonstrating significant potential for application in the discovery of clinical biomarkers.

Keywords:   N-Glycomics; HPLC-ESI-HRMS; Muscle atrophy; Phosphonium hydrazide; Simulated microgravity; Stable isotope relative quantitation

DOI:  https://doi.org/10.1007/s00216-026-06329-0
J Sep Sci. 2026 Jan;49(1): e70360

Quantitative Identification of Hydrolysis Products of Mustards by Novel Acetylation Method and Liquid Chromatography-High Resolution Mass Spectrometric Analysis.

Rama Krishna Maloth, Nagaraju Maddala, Swarajya Lakshmi Vattem Venkata, Satya Sri Dharmika Kanakala, Sai Krishna Ganji, Vijayasarathi Upadhyayula.

  A novel derivatization method was developed for the analysis of hydroxy degradation products of the Chemical Weapons Convention (CWC)-related chemicals present in aqueous and soil samples. The analytes were acetylated with acetyl chloride and quantitatively identified by liquid chromatography-tandem mass spectrometry technique in electrospray ionization and atmospheric pressure chemical ionization modes. The acetylated products showed good separation and peak shapes with characteristic fragmentation. The developed method was validated, and the results showed linearity in the range of 1-5000 ng/mL quantities from aqueous and soil samples. The intraday and interday validation results show the deviation of less than 10% with greater than 90% recovery. The limit of detection and limit of quantification of the method, measured at a signal-to-noise ratio of 3:1 and 10:1, respectively, are in the range of 1-20 ng/mL. The developed method was applied to the analysis of samples given in the 56th and 57th official proficiency tests conducted by the Organization for the Prohibition of Chemical Weapons (OPCW), and the chemicals were successfully identified by the developed method.

Keywords:  acetylation; aqueous samples; chemical warfare agents; environmental samples; liquid chromatography; mass spectrometry; soil samples

DOI:  https://doi.org/10.1002/jssc.70360
Bioanalysis. 2026 Jan 30. 1-10

Determination of R/S-enantiomers of methamphetamine and amphetamine in human hair with chiral stationary phase LC-MS/MS.

Yunfeng Zhang, Yong Liu, Chao Yuan, Weiwei Zhuge, Bo Zou, Linpei Dong, Xiaojun Wu, Zheng Ouyang, Manman Zhang, Haixing Wang.

   BACKGROUND: As the synthetic abused drug with high addictive potential, methamphetamine (METH) and its major metabolite amphetamine (AMP) are chiral compounds. The S-enantiomer of METH is primarily abused because of its potent psychoactive effects, whereas the R-enantiomer may originate from the metabolism of selegiline, a prescription medication for Parkinson's disease.
RESEARCH DESIGN AND METHODS: This research aimed to develop a robust and reliable analytical method to distinguish illicit METH abuse from legal selegiline therapy. A novel, simplified chiral stationary phase liquid chromatography-tandem mass spectrometry (CSP-LC-MS/MS) method was developed and validated for the rapid determination of R- and S-enantiomers of METH and AMP in human hair, eliminating the need for derivatization pretreatment.
RESULTS: Employing an Agilent Chiral-V column under isocratic conditions, the developed CSP-LC-MS/MS method achieved efficient baseline separation (resolution ≥2) and rapid quantification of the R/S enantiomers of METH and AMP within 10 min. Analysis of hair samples from three METH abusers revealed a predominance of the S-enantiomers. Conversely, only the R-enantiomer was detected in the hair of a selegiline user.
CONCLUSIONS: This research enables precise enantiomer differentiation, offering critical insights into drug metabolism and forensic discrimination between illicit METH abuse and legitimate selegiline treatment.

Keywords:  CSP-LC-MS/MS; R/S-amphetamine; R/S-methamphetamine; chiral analysis; hair; illicit drug abuse

DOI:  https://doi.org/10.1080/17576180.2026.2623033
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Jan 19. pii: S1570-0232(26)00015-2. [Epub ahead of print]1272 124926

An optimized HPLC-MS/MS assay for uracil and dihydrouracil in plasma: Improving dihydropyrimidine dehydrogenase phenotyping for individualized fluoropyrimidine treatment.

Yahia Bennani, Carla Chehade, Caroline Samer, Thibaud Kössler, Aurélien Thomas, Youssef Daali.

  Dihydropyrimidine dehydrogenase (DPD) is a key phase I drug-metabolizing enzyme responsible for catabolizing fluoropyrimidine chemotherapeutics such as 5-fluorouracil, capecitabine and tegafur. Its activity shows marked interindividual variability due to both genetic polymorphisms and environmental factors. Reduced DPD activity results in excessive fluoropyrimidine exposure, potentially causing severe and life-threatening toxicities. Although DPD testing is recommended before initiating fluoropyrimidine therapy, no single analytical strategy has been universally adopted, leading to substantial variability in clinical practice. While genotyping offers high specificity, phenotyping provides greater sensitivity by capturing both genetic and non-genetic influences on enzyme activity. In this study, we developed and validated an HPLC-MS/MS assay for the simultaneous quantification of endogenous uracil and dihydrouracil in human plasma, widely used endogenous biomarkers for DPD phenotyping. Sample preparation involved extraction with lipid removal, followed by chromatographic separation on porous graphitic carbon (100 × 2.1 mm, 5 μm; Hypercarb™, Thermo Scientific). Validation was performed according to international guidelines, demonstrating appropriate selectivity, sensitivity, linearity, accuracy, precision, carry-over, and stability. The method was applied to 28 human plasma samples, with uracil concentrations compared against an external laboratory using an independent LC-MS/MS platform. Deming regression showed no significant constant bias at the 95% confidence level, although underestimation occurred at higher concentrations (y = 0.63× + 4.86). Bland-Altman analysis indicated a small mean difference of -0.02 ng/mL but wide limits of agreement (-9.61 to +9.57 ng/mL), highlighting the impact of analytical variability near clinical decision thresholds. Further studies are warranted to evaluate the clinical utility of this approach in larger cohorts and to assess its correlation and complementarity with DPD genotyping and clinical outcomes. This method represents a valuable analytical tool to support personalized fluoropyrimidine-based chemotherapy in patients with solid tumors.

Keywords:  Dihydropyrimidine dehydrogenase; Dihydrouracil; Fluoropyrimidine; Phenotype; Uracil

DOI:  https://doi.org/10.1016/j.jchromb.2026.124926