bims-metlip 2025-12-28 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–12–28
29 papers selected by
Sofia Costa, Matterworks

Dual flow-path HPLC method for time-optimised quantification of physicochemically different metabolite classes in human serum samples using a single mass spectrometer.
A liquid chromatography-mass spectrometry method for profiling global phospholipid methylation via Headgroup analysis revealisng cell-type specificity.
A Roadmap guide on bioanalysis challenges and practical solutions for accurate quantification of oligonucleotide-based novel therapeutic modalities using LC-MS.
A Perspective on Unintentional Fragments and Their Impact on the Dark Metabolome, Untargeted Profiling, Molecular Networking, Public Data, and Repository Scale Analysis.
Local Sample Cohesion Normalization: Preserving Inherent Biological Heterogeneity in Metabolomics Data.
Fast quantification of methylated nucleosides in urine by triple quadrupole mass spectrometry.
An Enhanced and Highly Sensitive LC-MS-Based Analytical Approach for the Quantitative Determination of Carcinogenic N-Nitrosopiperazine in Cetirizine Dihydrochloride.
Multiresidue determination of alkylamines and alkylamides in meat by liquid chromatography high-resolution mass spectrometry and implications for food safety.
HGAlign: Biologically preserving batch correction and classification for metabolomics via heterogeneous graph alignment.
Streamlining feature elaboration and statistics analysis in metabolomics: the GetFeatistics R-package.
Biomonitoring of Environmental Phenols, Phthalate Metabolites, Triclosan, and Per- and Polyfluoroalkyl Substances in Humans with Chromatography and Mass Spectrometry.
A Comprehensive Review of Analytical Techniques for Doxycycline and Its Metabolites.
Advanced Tissue Imprinting With Pneumatic Press for Mass Spectrometry Imaging of Plant Tissues.
Gamma-hydroxybutyric acid (GHB) derivatization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC): LC-HRAM-Orbitrap-MS characterization of GHB and deuterated GHB derivatives and application to blood, urine, and hair analysis.
UHPLC Method Development for the Trace Level Quantification of Anastrozole and Its Process-Related Impurities Using Design of Experiments.
Creating taxonomically-informed metabolome libraries for any species using the pubchem.bio R package.
Overcoming Analytical Challenges for the Detection of 27 Cyanopeptides Using a UHPLC-QqQ-MS Method in Fish Tissues.
A novel 1H NMR-based quantitative method for the direct quantification of mono-rhamnolipids and di-rhamnolipids.
Kendrick Mass Defect Filtering Enables High-Throughput Untargeted Annotation of Minor Phytocannabinoids: Toward Streamlined Phytocannabinomics.
Improving sensitivity and selectivity of human ACTH[1-39] quantitation using online size exclusion chromatography and antibody-free LC-HRMS.
New Multiplex LC-MS/MS Method for lipid biomarker Analysis of Inherited Neurodegenerative Metabolic Diseases.
Fragmentation Patterns of Chlorogenic Acid Homologs in Positive- and Negative-Ion Mode Mass Spectrometry.
Spatial Ion Compression without Loss of Resolution on a Cyclic Ion Mobility Spectrometry-Mass Spectrometry Platform.
Chiral Derivatization Enables High-Resolution Ion Mobility Spectrometry of 30 Amino Acid Enantiomers.
Analytical Techniques for Quantification of Metoclopramide: A Comprehensive Review.
Determination of paraquat in drinking water by HPLC-UV using large-volume injection and a superficially porous particle HILIC column.
Screening of active ingredients from traditional Chinese medicine using two-dimensional liquid chromatography-mass spectrometry: A case study of screening neuraminidase inhibitors from Schizonepetae spica.
Development and validation of a rapid LC-MS/MS method for methylphenidate, atomoxetine, and their metabolites with application to pediatric pharmacokinetics.
Early Detection of Infectious Diseases: A Review of Recent Advances in Pathogen Identification, Molecular Tools, and Metabolomics-Driven Biomarker Discovery.

J Chromatogr A. 2025 Dec 17. pii: S0021-9673(25)00980-X. [Epub ahead of print]1767 466636

Dual flow-path HPLC method for time-optimised quantification of physicochemically different metabolite classes in human serum samples using a single mass spectrometer.

Christian Faist, Edda Tacke, Thorsten Eierhoff, Alexander Oberhuber, Heiko Hayen.

  Biomedical studies often involve the analysis of a wide range of analytes. The varying physicochemical properties of these analytes frequently hinder their detection by a single method, making it necessary to employ different analytical instruments or chromatographic approaches. This process often requires considerable time. In this study, we present a time-optimised LC-MS-based approach using only one mass spectrometer, but utilisation of two orthogonal chromatographic techniques, namely hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP)-HPLC in parallel. The established method allowed short-chain fatty acids (SCFA) and amines (such as trimethylamine N-oxide and carnitine) to be recovered from one human serum sample. SCFA were derivatized to their corresponding 3-nitrophenylhydrazones for improved RP-HPLC separation and electrospray ionisation efficiency, whereas amines could be separated and detected in their native forms on a zwitterionic HILIC phase. The combination of RP and HILIC methods through a six-port valve was achieved with a dual split sampler, enabling re-equilibration of one chromatographic column during the separation step of the other method. This approach ensured optimal capacity use of the MS and resulted in a 37 % analysis time reduction compared to the analysis with both individual methods. Application to human serum samples confirmed the robustness and reproducibility of the method, with quantification results showing relative standard deviations ≤ 5.8 % for all analytes as well as stable retention times and chromatographic resolutions for both methods. By allowing the combination of any chromatographic columns and methods with only one detector, this setup is well suited for quantification of extensive sample batches applying customised chromatographic conditions, while ensuring the optimal separation performance for both methods as well as maintaining efficient detector utilisation.

Keywords:  LC-MS; Microbiota; Parallelised chromatography; Quantification; Short-chain fatty acid; Trimethylamine N-oxide

DOI:  https://doi.org/10.1016/j.chroma.2025.466636
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Dec 17. pii: S1570-0232(25)00457-X. [Epub ahead of print]1270 124903

A liquid chromatography-mass spectrometry method for profiling global phospholipid methylation via Headgroup analysis revealisng cell-type specificity.

Hongjun Luo, Hui Li, Yongyin Zhou, Wenhong Luo, Zhexuan Lin.

  Global analysis of phospholipid methylation is crucial for understanding the biological roles of phospholipids in mammalian membranes. This study developed and validated a robust liquid chromatography-mass spectrometry (LC-MS) method for the simultaneous determination of ethanolamine (EA) and choline (CL) released from the hydrolysis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), respectively. The extraction, hydrolysis, and detection procedures were optimized. The compounds were separated on an HILIC column with isocratic elution and detected using a mass spectrometer with electrospray ionization in positive ion mode. The selected ions monitored were m/z 104.1 for CL, m/z 113.1 for d9-CL (internal standard for CL), m/z 62.1 for EA, and m/z 66.1 for d4-EA (internal standard for EA). The method showed satisfactory linearity (r2 > 0.999), precision (intra- and inter-day RSDs ≤6.3 %), and accuracy (-2.1-6.8 %). The limits of detection (LOD) were 0.30 μM for EA and 0.02 μM for CL. This method was applied to human erythrocytes and several tumor cell lines (Hela, EC109, Saos-2), revealing significant differences in the EA/CL ratio (reflecting the PE/PC ratio) among different cell types. This study indicated that the global phospholipid methylation status is cell-type specific and established the approach as a valuable tool for investigating membrane lipid composition in health and disease.

Keywords:  Choline; Ethanolamine; LC-MS; Phosphatidylcholine; Phosphatidylethanolamine; Phospholipids

DOI:  https://doi.org/10.1016/j.jchromb.2025.124903
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Dec 17. pii: S1570-0232(25)00454-4. [Epub ahead of print]1270 124900

A Roadmap guide on bioanalysis challenges and practical solutions for accurate quantification of oligonucleotide-based novel therapeutic modalities using LC-MS.

Devendra Kumar, Manish Sharma, Neerja Trivedi.

  Therapeutic oligonucleotides have emerged as a transformative drug class, yet their physicochemical complexity poses unique analytical challenges in bioanalysis. Liquid chromatography mass spectrometry (LC-MS) has become a powerful platform for their quantification, offering high specificity and structural insight. However, accurate measurement requires addressing challenges such as nonspecific binding, matrix effects, nuclease degradation, and ion-pairing interferences from sample preparation to LC-MS analysis. This review provides a practical roadmap for establishing robust LC-MS workflows for oligonucleotides bioanalysis, emphasizing optimized sample preparation, column and mobile phase selection, ionization control, and fragmentation tuning. Key strategies to minimize analytical artifacts, improve recovery, and ensure regulatory compliance are discussed in the context of current FDA or EMA bioanalytical validation guidelines. Collectively, this work outlines the critical considerations and systematic optimizations needed to achieve reliable, reproducible, and sensitive quantification of therapeutic oligonucleotides in complex biological matrices, supporting their successful clinical translation by informing pharmacokinetics, therapeutic potential, and safety profiles.

Keywords:  Columns; Ion-pairing reagents; LC-MS/MS; Non-Specific Binding; Oligonucleotides; Pharmacokinetics; SPE

DOI:  https://doi.org/10.1016/j.jchromb.2025.124900
JACS Au. 2025 Dec 22. 5(12): 5828-5850

A Perspective on Unintentional Fragments and Their Impact on the Dark Metabolome, Untargeted Profiling, Molecular Networking, Public Data, and Repository Scale Analysis.

Yasin El Abiead, Ipsita Mohanty, Shipei Xing, Adriano Rutz, Vincent Charron-Lamoureux, Tito Damiani, Wenyun Lu, Gary J Patti, Nicola Zamboni, Oscar Yanes, Pieter C Dorrestein.

  In/postsource fragments (ISFs) arise during electrospray ionization or ion transfer in mass spectrometry when molecular bonds break, generating ions that can complicate data interpretation. Although ISFs have been recognized for decades, their contribution to untargeted metabolomicsparticularly in the context of the so-called "dark matter" (unannotated MS or MS/MS spectra) and the "dark metabolome" (unannotated molecules)remains unsettled. This ongoing debate reflects a central tension: while some caution against overinterpreting unidentified signals lacking biological evidence, others argue that dismissing them too quickly risks overlooking genuine molecular discoveries. These discussions also raise a deeper question: what exactly should be considered part of the metabolome? As metabolomics advances toward large-scale data mining and high-throughput computational analysis, resolving these conceptual and methodological ambiguities has become essential. In this perspective, we propose a refined definition of the "dark metabolome" and present a systematic overview of ISFs and related ion forms, including adducts and multimers. We examine their impact on metabolite annotation, experimental design, statistical analysis, computational workflows, and repository-scale data mining. Finally, we provide practical recommendationsincluding a set of dos and do nots for researchers and reviewersand discuss the broader implications of ISFs for how the field explores unknown molecular space. By embracing a more nuanced understanding of ISFs, metabolomics can achieve greater rigor, reduce misinterpretation, and unlock new opportunities for discovery.

Keywords:  analytical artifact; dark metabolome; electrospray ionization; in-source fragmentation; mass spectrometry; metabolomics

DOI:  https://doi.org/10.1021/jacsau.5c01063
Anal Chem. 2025 Dec 23.

Local Sample Cohesion Normalization: Preserving Inherent Biological Heterogeneity in Metabolomics Data.

Fanjing Guo, Lingli Deng, Kian-Kai Cheng, Keyi Lu, Yongpei Wang, Lei Guo, Daniel Raftery, Jiyang Dong.

Metabolomics data from biofluids like urine or cell cultures are frequently confounded by unwanted overall sample concentration (or dilution effects). Conventional normalization methods, such as Constant Sum Normalization (CSN), L2-Norm Normalization (L2N), Probabilistic Quotient Normalization (PQN), and Quantile Normalization (QT), rely on a unified global reference, failing to account for inherent biological heterogeneity (e.g., interindividual variability, subgroup divergences). This limitation can distort biological data structures and compromise downstream analyses. To address this issue, we developed Local Sample Cohesion Normalization (LSCN), that corrects dilution effects while preserving biological heterogeneity. LSCN constructs a sample-specific neighbor set for each spectrum based on pairwise similarity in a reduced-dimensional space and performs locally weighted normalization within these neighborhoods. This approach mitigates technical bias without enforcing artificial global uniformity. We rigorously validated LSCN against CSN, L2N, PQN, and QT normalization using simulated data sets with known heterogeneity and diverse real-world metabolomics data sets (urine, cells, tissues, tea leaves). LSCN demonstrated superior performance in Preserving heterogeneity, achieving significantly higher global and local structural similarity to ground-truth references; Retaining biological signals, enhancing identification of differential metabolites, correlation networks, pathway enrichment, and classification accuracy; and Effectively correcting dilution effects, yielding more accurate normalization factors and reduced within-group variance. LSCN offers a robust, biologically faithful preprocessing framework for metabolomics, improving reliability in downstream analyses.

DOI: https://doi.org/10.1021/acs.analchem.5c04710
Anal Biochem. 2025 Dec 23. pii: S0003-2697(25)00275-1. [Epub ahead of print] 116036

Fast quantification of methylated nucleosides in urine by triple quadrupole mass spectrometry.

Carmen Mena-Iglesias, Myriam Bustamante-Rangel, Diego García-Gómez, M Teresa Fernández-Del-Campo-García, Encarnación Rodríguez-Gonzalo, José Luis Pérez Pavón.

  Modified nucleosides and nucleobases have raised great scientific interest due to their possible use as diagnostic and prognostic markers for various diseases. One of the most common types of modification is methylation. Sensitive and accurate methods are essential for the quantification of these compounds in biological samples. In this work we propose, for the first time, a rapid non-separative method for the determination of methylated nucleosides in urine by direct injection on electrospray ionization triple quadrupole mass spectrometer (ESI-MS/MS). The proposed method has been applied to the simultaneous determination of five methylated nucleosides in human urine samples. A separative method based on HILIC-MS/MS has been developed in parallel with the same instrumental setup to confirm the results obtained by the non-separative method. Both methods were validated in terms of linearity, matrix effect, limits of quantification, precision and recovery. The proposed method was successfully applied to the determination of the selected analytes in the urine of healthy volunteers and was compared with the results obtained by the separative method. The proposed non-separative method considerably reduced the required analysis time and simplified the sample treatment, which is a remarkable advantage over the chromatographic methods described. Therefore, this method is suitable for rapid, simple, and reliable determination of endogenous biomarkers in urine samples for routine or high-throughput analysis. The separative method is recommended as a complementary tool for confirmation in complex situations or in cases of abnormally high biomarker levels, maintaining specificity and robustness without compromising the practical advantages of the rapid method.

Keywords:  ESI-MS/MS; biomarker; methylated nucleosides; non-separative method

DOI:  https://doi.org/10.1016/j.ab.2025.116036
Biomed Chromatogr. 2026 Feb;40(2): e70336

An Enhanced and Highly Sensitive LC-MS-Based Analytical Approach for the Quantitative Determination of Carcinogenic N-Nitrosopiperazine in Cetirizine Dihydrochloride.

Mithun M Gharat, Pallavi T Roy, Ramchandra G Thorat, Amit N Gosar, Tabrez A Shaikh, Nitin A Mirgane.

  Nitrosamine impurities, which are categorized as mutagenic and probable human carcinogens, have been widely detected in various drug substances and products. This study investigates the formation of the N-nitrosopiperazine (NNPZ) genotoxic impurity during the manufacturing of cetirizine dihydrochloride (CTZ), originating from piperazine, a key starting material. A sensitive and selective LC-MS method was developed for the accurate quantification of NNPZ in the CTZ drug substance. The allowable intake limit for NNPZ is 26.5ng/day and maximum daily dose 880mg/day as per US FDA. This analytical method was developed and validated in accordance with ICH Q2R2 guidelines. It demonstrated a low detection limit (LOD) of 0.03 ng/mL and a quantitation limit (LOQ) of 0.1 ng/mL. The linearity study yielded a strong regression coefficient of 0.9974. With a high recovery rate of 96.56% to 102.35% for spiked NNPZ, the method's accuracy was well-established. The overall results confirm that this methodology is reliable, precise, and reproducible, and it can therefore be used for the determination of NNPZ-genotoxic impurity in CTZ drug substance samples via LC-MS.

Keywords:  N‐nitrosopiperazine (NNPZ); analytical method validation; cetirizine dihydrochloride (CTZ); liquid chromatography–mass spectrophotometer (LC–MS); nitrosamine

DOI:  https://doi.org/10.1002/bmc.70336
J Chromatogr A. 2025 Dec 19. pii: S0021-9673(25)00986-0. [Epub ahead of print]1768 466642

Multiresidue determination of alkylamines and alkylamides in meat by liquid chromatography high-resolution mass spectrometry and implications for food safety.

Ling Liu, Yizhe Zhu, Zitao Chai, Ruifen Zheng, Zhiqiang Zeng, Caiming Tang.

  This study developed a rapid and reliable liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for the simultaneous determination of 8 alkylamines and 12 alkylamides in meat products. Sample preparation and instrumental conditions were optimized to achieve efficient extraction and accurate quantification. Meat samples were extracted with methanol-acetonitrile mixture (1:1, v/v) using vortex-ultrasonication for 15 min, followed by evaporation and reconstitution prior to analysis. The mobile phases were ultrapure water containing 0.1 % formic acid and acetonitrile, and a gradient elution program was employed. Mass spectrometric analysis was performed in positive electrospray ionization mode with full scan and data-dependent acquisition. Quantification was carried out using an internal standard method. The limits of quantification were 5 ng/g, and calibration curves showed good linearity. Recoveries ranged from 85.2 % to 103.0 % with relative standard deviations (RSDs) of 0.3-8.0 %, and matrix effects ranged from 61.7 % to 109.6 % with RSDs of 0.2-10.3 %. The method was successfully applied to meat samples collected from local markets, with concentrations of individual alkylamines and alkylamides ranging from below the detection limit to 13,105.0 ng/g. Migration experiments indicated that alkylamines and alkylamides could transfer from plastic materials into meat, with migration extent increasing with the increase of contact time and temperature. Hydrophobic alkylamides migrated more rapidly in early stage, while some alkylamines continued to migrate at low temperatures, indicating that meat characteristics and contact conditions could significantly affect the migration. The developed method is suitable for the determination of alkylamines and alkylamides in meat products, and can provide a reference approach for assessing the potential migration and exposure risks of alkylamines and alkylamides.

Keywords:  Alkylamides; Alkylamines; Food safety; Liquid chromatography high-resolution mass spectrometry; Meat

DOI:  https://doi.org/10.1016/j.chroma.2025.466642
Comput Biol Chem. 2025 Dec 19. pii: S1476-9271(25)00528-6. [Epub ahead of print]121 108864

HGAlign: Biologically preserving batch correction and classification for metabolomics via heterogeneous graph alignment.

Yang Gao, Haoyun Yu, Chunman Zuo.

  Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) is a powerful tool for profiling complex biological samples. However, large-scale metabolomics experiments often suffer from substantial batch effects caused by variations in sample processing, instrument conditions, and acquisition protocols. These non-biological variations obscure true biological signals, reduce reproducibility, and compromise the generalizability of downstream models. Existing correction methods either rely on oversimplified linear assumptions or risk over-correcting and removing meaningful biological differences. To address this challenge, we propose HGAlign(Heterogeneous Graph Alignment Model), a neural network model that corrects batch effects in large-scale MALDI-MS experiments while preserving important biological differences. Our approach uses heterogeneous graph convolutional networks to learn relationships between samples and metabolic features, enabling effective batch correction without losing disease-related information. Extensive experiments on CyTOF public datasets and clinical MALDI-MS serum data from systemic lupus erythematosus (SLE) patients demonstrate that HGAlign significantly reduces inter-batch discrepancies while maintaining or improving classification accuracy. Quantitative evaluation shows that our method achieves the lowest MMD values among state-of-the-art methods, and consistently improves classification metrics. Moreover, HGAlign avoids over-correction, enabling stable identification of cross-batch differential metabolites that retain biological interpretability. HGAlign offers a principled framework for balancing batch effect removal and biological signal preservation in high-throughput metabolomics. By introducing heterogeneous graph representation learning, it achieves superior performance in both batch correction and disease classification tasks, showing strong potential for large-scale clinical applications.

Keywords:  Batch correction; Deep learning; Heterogeneous graph convolution; Metabolomics

DOI:  https://doi.org/10.1016/j.compbiolchem.2025.108864
J Integr Bioinform. 2025 Dec 24.

Streamlining feature elaboration and statistics analysis in metabolomics: the GetFeatistics R-package.

Gianfranco Frigerio.

  Metabolomics studies require complex data processing pipelines to ensure data quality and extract meaningful biological insights. GetFeatistics is an R-package developed to streamline the elaboration and statistical analysis of metabolomics data. For targeted analyses, the package enables calibration curve-based quantification with different data weighting options. For untargeted studies, it includes dedicated functions to import feature tables from tools like patRoon and MS-DIAL, assign annotation confidence levels, and filter features based on pooled quality control (QC) criteria, including options for group-specific pooled QCs. The package also provides functions for univariate and multivariate statistical analyses, notably streamlined regression modelling with fixed effects, mixed-effects models for longitudinal data, and Tobit regression for censoring values exceeding the limits of detection. Output tables are concise and informative, facilitating interpretation and reporting, while output visualisations are fully customisable via the ggplot grammar. Additional functionalities include automated retrieval of chemical properties from PubChem, ontology classification via ClassyFire, and pathway enrichment analysis using the FELLA package. GetFeatistics is publicly available on GitHub, with comprehensive documentation and a step-by-step vignette. By integrating key steps of the metabolomics workflow, the package aims to facilitate both exploratory studies and large-scale epidemiological applications in metabolomics research.

Keywords:  computational metabolomics; data preprocessing; metabolomics workflow; non-targeted metabolomics; open-source software

DOI:  https://doi.org/10.1515/jib-2025-0047
Toxics. 2025 Nov 28. pii: 1029. [Epub ahead of print]13(12):

Biomonitoring of Environmental Phenols, Phthalate Metabolites, Triclosan, and Per- and Polyfluoroalkyl Substances in Humans with Chromatography and Mass Spectrometry.

Anastasia Chrysovalantou Chatziioannou, Antonis Myridakis, Euripides G Stephanou.

  Endocrine-disrupting chemicals (EDCs) are widespread organic compounds that interfere with hormone signaling and are linked to reproductive, developmental, cardiovascular, and cancer-related health effects. Key EDCs include bisphenol-A and its analogs, phthalates, parabens, triclosan, and per-and polyfluoroalkyl substances (PFAS), which are commonly present in personal care products and plastics. Human exposure occurs via environmental exposure through ingestion, inhalation, and dermal contact, with persistent compounds such as PFAS accumulating in blood, while others are excreted in urine as free or conjugated metabolites. Accurate assessment of EDC exposure, particularly during pregnancy and early childhood, requires robust analytical methods. Liquid and gas chromatography coupled with mass spectrometry (LC-MS and GC-MS) are the most widely used techniques to date. LC-MS is favored for its sensitivity, specificity, and minimal sample preparation, whereas GC-MS provides adequate performance but often requires time-consuming derivatization. This review summarizes current LC-MS and GC-MS methodologies for multi-class EDC biomonitoring, emphasizing sample preparation, analyte coverage, and methodological strengths and limitations, providing a practical reference for human exposure studies using common biological matrices such as urine and blood.

Keywords:  biomonitoring; chemical analysis; chromatography; endocrine disruptors; human biofluids; mass spectrometry

DOI:  https://doi.org/10.3390/toxics13121029
Biomed Chromatogr. 2026 Feb;40(2): e70322

A Comprehensive Review of Analytical Techniques for Doxycycline and Its Metabolites.

Hemn A H Barzani, Rebaz Anwar Omer, Nergz Bayiz Abdulrahman, Zanco Hassan Jawhar, Seerwan Hamadameen Sulaiman.

  Doxycycline (DC), a second-generation tetracycline antibiotic, is extensively used in human and veterinary medicine for its broad-spectrum activity, favorable pharmacokinetics, and high bioavailability. This review focuses on analytical methods for detecting and quantifying DC and its metabolites in pharmaceutical, biological, and environmental samples. Spectroscopic, chromatographic, and electroanalytical techniques have been explored, with high-performance liquid chromatography (HPLC) emerging as the most reliable due to its high sensitivity, selectivity, and reproducibility, particularly when coupled with fluorescence or mass spectrometric detectors. Although spectroscopic approaches are simple and economical, they often suffer from matrix interferences. Electroanalytical and voltammetric methods, by contrast, offer rapid and cost-effective alternatives suitable for field applications. Major analytical challenges include DC instability, complex sample matrices, and intricate preparation steps affecting quantification accuracy. Future research should focus on green, miniaturized, and automated analytical platforms that integrate nanomaterials, advanced separation techniques, and artificial intelligence to achieve rapid, accurate, and sustainable monitoring of DC and its metabolites in clinical and environmental settings.

Keywords:  analytical techniques; chromatography; doxycycline; drug quantification; electrochemical analysis; environmental monitoring; metabolites; spectroscopy

DOI:  https://doi.org/10.1002/bmc.70322
J Mass Spectrom. 2026 Jan;61(1): e70007

Advanced Tissue Imprinting With Pneumatic Press for Mass Spectrometry Imaging of Plant Tissues.

Pubudu Nuwan Perera Hapuarachchige, Vy T Tat, Young Jin Lee.

Sample preparation is an important first step to obtain high quality mass spectrometry imaging (MSI) data. Preparing plant tissues is especially challenging for MSI of thin tissues along the lateral dimensions. The unique challenges involved with plant tissues, such as fragile cell walls, hydrophobic barriers, and specific tissue structures, often lead to inefficiency and difficulties in sample preparation. Imprinting plant tissues onto porous polytetrafluoroethylene (pPTFE) sheet has been widely used to extract internal metabolites in leaves and petals while keeping spatial resolution for MSI. However, pressure applications were typically made manually using a vise or pliers leading to low reproducibility and resolution in MS images. In this study, we introduce a home-built pneumatic press (PNP) that has been designed to precisely control the pressure application parameters during imprinting. To evaluate the performance of the new device, Lemna minor fronds, Arabidopsis thaliana, and Bacopa monnieri leaves were imprinted onto the pPTFE with PNP, vise, or pliers, and matrix-assisted laser desorption/ionization (MALDI) MSI was obtained on the imprints. The PNP showed dramatic improvements in reproducibility and image quality compared to manual pressure application tools.

DOI: https://doi.org/10.1002/jms.70007
Anal Methods. 2025 Dec 22.

Gamma-hydroxybutyric acid (GHB) derivatization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC): LC-HRAM-Orbitrap-MS characterization of GHB and deuterated GHB derivatives and application to blood, urine, and hair analysis.

Giampietro Frison, Arianna Negro, Flavio Zancanaro, Samuela Frasson, Gianpaola Tedeschi, Camilla Vigato, Giorgia Miolo.

This paper describes a new analytical approach for determining gamma-hydroxybutyric acid (GHB) in biosamples using liquid chromatography-mass spectrometry (LC-MS) following derivatization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC). The derivatization procedure is simple, rapid, reproducible, inexpensive, and safe. It allows for a complete variation of GHB chemical characteristics (from hydroxyacid to N-acylurea) with a molecular weight increase from 104 to 259 u and the introduction of 3 nitrogen atoms. These modifications promote the protonation of the analytes in the MS electrospray ion source, enabling MS detection under positive ionization conditions. Liquid chromatography-high-resolution accurate-mass Orbitrap mass spectrometry (LC-HRAM-Orbitrap-MS) measurements demonstrated that derivatization produces two GHB-EDC derivatives with the same exact mass (MH+ ions at m/z 260.1968) and experimental isotopic patterns overlapping each other and superimposable to the calculated one. Thus, they share an identical elemental composition (C12H25O3N3) but have different molecular structures (GHB-EDCA and GHB-EDCB). Equivalent results were obtained for D6-GHB: the production of two deuterated N-acylureas (D6-GHB-EDCA and D6-GHB-EDCB), with MH+ ions at m/z 266.2341, superimposable experimental and calculated isotopic patterns, elemental composition C12H19D6O3N3, and different molecular structures, mirroring those of GHB-EDCA and GHB-EDCB. After optimizing the derivatization conditions (reaction solvent, reaction temperature and time, and volume and concentration of the derivatizing agent) the final procedure involves reacting with 10 mM aqueous EDC, at 45 °C for 15 minutes. GHB-EDC derivatives were found to be highly stable over time (at least 15 days), even at room temperature. Three preliminary analytical methods for the determination of endogenous and exogenous GHB levels in urine, blood, and hair samples were developed.

DOI: https://doi.org/10.1039/d5ay01461a
Biomed Chromatogr. 2026 Feb;40(2): e70279

UHPLC Method Development for the Trace Level Quantification of Anastrozole and Its Process-Related Impurities Using Design of Experiments.

Narasimha Rao Tadisetty, Ramesh Yanamandra, Subhalaxmi Pradhan, Gitanjali Pradhan.

  A sensitive, selective, and robust ultra-high-performance liquid chromatography method was developed for the simultaneous quantification of anastrozole and five process-related impurities at trace levels (≤ 0.1%). Traditional univariate optimization was replaced with a systematic chemometric approach using Box-Behnken Design to evaluate critical chromatographic parameters-mobile phase pH (3.0-4.0), column temperature (25°C-35°C), and flow rate (0.20-0.60 mL/min). Response Surface Methodology was employed to model retention behavior, resolution, and peak symmetry, with desirability function optimization identifying the ideal conditions (pH 3.20, 29.3°C, 0.33 mL/min). The method achieved baseline separation on an ACQUITY BEH C18 column (2.1 × 50 mm, 1.7 μm) using 10-mM ammonium formate (pH-adjusted) and acetonitrile as the mobile phase. Validation per International Council for Harmonisation Q2(R1) guidelines confirmed excellent linearity (R2 ≥ 0.999), precision (%RSD ≤ 1.5% for ANZ; ≤ 5.3% for impurities), accuracy (recoveries: 94%-101%), and sensitivity (limit of detection: 0.011-0.014 μg/mL; limit of quantitation: 0.020-0.025 μg/mL). This study reports the first validated ultra-high-performance liquid chromatography method enabling trace-level quantification of two newly identified (IMP-1 and IMP-2) and three previously reported process-related impurities of anastrozole in a single run, using a Design of Experiments-based optimization approach.

Keywords:  Box–Behnken Design; Design of Experiments; UHPLC method development; anastrozole; method validation; process‐related impurities; trace‐level quantification

DOI:  https://doi.org/10.1002/bmc.70279
Analyst. 2025 Dec 24.

Creating taxonomically-informed metabolome libraries for any species using the pubchem.bio R package.

Corey D Broeckling.

Annotation remains a significant challenge in metabolomics, in large part due to the enormous structural diversity of small molecules. PubChem represents one of the largest curated chemical structure databases, with more than 122 000 000 structures, supplemented by extensive biological metadata provided by numerous external sources. While many of these structures are relevant to metabolomics, a majority are unlikely to be measured in a typical metabolomics experiment. This article describes the R package, pubchem.bio, which enables users to: (1) download the metabolomics-centric subset of PubChem onto their local computer, (2) build a metabolomic structured library of biological compounds in PubChem, (3) develop custom metabolite structure libraries for any species or collection of species using selected or all available taxonomic data in PubChem and (4) define a core biological metabolome, comprising metabolites plausibly found in any species. Species-specific metabolomes are enabled through the adoption of a lowest-common-ancestor chemotaxonomy approach, which is implemented by associating PubChem CIDs into the NCBI Taxonomy database hierarchy, enabling extrapolation of the taxonomic range beyond the species reported. This package is available via CRAN, and can be used to simplify the annotation process and embed biological metadata into the annotation process.

DOI: https://doi.org/10.1039/d5an00914f
Toxins (Basel). 2025 Dec 02. pii: 580. [Epub ahead of print]17(12):

Overcoming Analytical Challenges for the Detection of 27 Cyanopeptides Using a UHPLC-QqQ-MS Method in Fish Tissues.

Audrey Roy-Lachapelle, François-Xavier Teysseire, Christian Gagnon.

  The increasing occurrence of harmful cyanobacterial blooms in freshwater ecosystems poses important risks to aquatic organisms and human health due to the production of bioactive secondary metabolites such as cyanopeptides. While analytical methods for microcystins are well developed, there is a notable lack of validated protocols for the broader spectrum of cyanopeptides in biota. This study presents the development and validation of a robust UHPLC-QqQ-MS method for the simultaneous extraction, cleanup, and quantification of 27 cyanopeptides, including microcystins, anabaenopeptins, microginins, aeruginosins, aeruginoguanidine, and nodularin, in fish muscle, liver, and whole fish tissues. Comprehensive optimization was conducted to minimize matrix effects and analyte losses during every step of sample preparation. The method demonstrated generally high recoveries (28-98%), good precision (RSD < 20%), and sensitivity, with MQLs below 0.5 ng g-1 for most analytes. Microginins posed analytical challenges due to their amphiphilic structure, which contributed to significant losses during filtration and extraction; the reasoning is discussed. Application to wild fish collected after a mass mortality event revealed no detectable cyanopeptide contamination but confirmed the method's suitability for comprehensive detection. This represents an important advancement in cyanopeptide analysis, offering a valuable tool for environmental risk assessment and food safety evaluation related to harmful cyanobacteria.

Keywords:  UHPLC-QqQ-MS; aeruginosins; anabaenopeptins; cyanobacteria; cyanopeptides; cyanotoxins; fish tissue analysis; harmful cyanobacterial blooms; microcystins; microginins

DOI:  https://doi.org/10.3390/toxins17120580
Talanta. 2025 Dec 24. pii: S0039-9140(25)01791-6. [Epub ahead of print]301 129300

A novel 1H NMR-based quantitative method for the direct quantification of mono-rhamnolipids and di-rhamnolipids.

Dongmei Li, Xianfa Lan, Weiyi Tao, Junzhang Lin, Zihui Chen, Shuang Li.

  Rhamnolipids are environmentally friendly biosurfactants with a wide range of applications; however, their structural complexity and the limited availability of high-purity standards pose significant analytical challenges. In this study, we developed a 1H NMR-based method for the direct quantification of both mono-rhamnolipids and di-rhamnolipids within complex mixtures. By identifying characteristic proton signals and optimizing a mixed deuterated solvent system (DMSO‑d6/CD3OD, 4:1 v/v), accurate quantification was achieved using sodium p-hydroxybenzoate as an internal standard. The method demonstrated strong anti-interference capability, excellent reproducibility (RSD <2 %), high accuracy (relative error <5 %), a relative abundance detection limit of 5 %, and satisfactory spike recovery rates, thereby addressing the limitations of conventional analytical techniques. This approach eliminates the need for high-purity reference standards and simplifies sample preparation, providing a reliable and efficient tool for both rhamnolipids research and industrial quality control.

Keywords:  (1)H NMR; Biosurfactants; Quantitative analysis; Rhamnolipids

DOI:  https://doi.org/10.1016/j.talanta.2025.129300
ACS Meas Sci Au. 2025 Dec 17. 5(6): 912-922

Kendrick Mass Defect Filtering Enables High-Throughput Untargeted Annotation of Minor Phytocannabinoids: Toward Streamlined Phytocannabinomics.

Andrea Cerrato, Giuseppe Cannazza, Cinzia Citti, Aldo Laganà, Roberta Paris, Anna Laura Capriotti.

  Phytocannabinoids are a diverse class of bioactive compounds produced by Cannabis sativa, including both major and a growing number of minor constituents with pharmacological relevance. However, their comprehensive annotation in untargeted high-resolution mass spectrometry (HRMS) data sets remains a significant analytical challenge due to their structural similarity, low abundance, and the complexity of plant matrices. In this study, we present a comparative evaluation of Kendrick Mass Defect (KMD)-based filtering workflows for the efficient untargeted annotation of minor phytocannabinoids. Three data processing strategies were implemented using Compound Discoverer: (i) KMD filtering before the "Compound Detection" tool, (ii) KMD filtering after the "Compound Detection" tool, and (iii) a pseudo-KMD approach based on the generation of expected compounds. These workflows were tested and compared using a data set comprising 50 Cannabis inflorescence samples analyzed in an untargeted fashion, taking into account the phytocannabinoid coverage, false positive rates, computation burden, and versatility. A total of 61 phytocannabinoids were annotated, including a full series of alkyl homologues (C1-C7), cis/trans isomers, O-methylated derivatives, and sesquicannabinoids. Statistical analyses revealed meaningful chemical differentiation based on seed origin, chemovar classification, and reproductive strategy (dioecious vs monoecious), highlighting the biological significance of minor cannabinoids. Overall, the results demonstrate that KMD filtering significantly enhances the throughput and accuracy of untargeted HRMS workflows for structurally related classes of compounds.

Keywords:  C. sativa; cannabinoids; chemovar; compound discoverer; geographical origin; high-resolution mass spectrometry

DOI:  https://doi.org/10.1021/acsmeasuresciau.5c00106
Bioanalysis. 2025 Dec 26. 1-9

Improving sensitivity and selectivity of human ACTH[1-39] quantitation using online size exclusion chromatography and antibody-free LC-HRMS.

B R Jones, L Shan, A Spytko, R Luo, A Ayala, J Wang, S Lowes.

   BACKGROUND: Quantitation of adrenocorticotropic hormone (ACTH) in human plasma by clinical immunoassays is prone to selectivity challenges, whereas quantitation by mass spectrometry assays (MSA) is limited by sensitivity. The use of nano-liquid chromatography (nano-LC) addresses the MSA sensitivity gap but commonly suffers from poor robustness due to LC system backpressure issues, which is mitigated by incorporation of online size exclusion chromatography (SEC).
RESEARCH DESIGN AND METHODS: An antibody-free, multidimensional nano-LC, high-resolution mass spectrometry method was designed and implemented to measure intact ACTH[1-39] in human plasma. Online SEC separation was performed at relatively high flow rates prior to trap and elute reversed phase separations at sub-µL/min flow rates. ACTH[1-39] was detected using a high-resolution orbitrap mass analyzer.
RESULTS AND CONCLUSIONS: Nanoelectrospray allows measurement of ACTH[1-39] to a clinically relevant concentration of 3-5 pg/mL, with low bias and high precision across the concentration range interrogated. SEC reduces microfluidic backpressure effects commonly observed with the use of nano-LC for bioanalytical applications. Eliminating antibody capture reduces susceptibility to endogenous antibody interferences. This format can be readily adapted for multiplexed measurement of other low abundance peptide hormones in biological samples.

Keywords:  ACTH; LC-MS; critical reagent-free; multi-dimensional LC separation; peptide quantitation; robustness; selectivity; sensitivity

DOI:  https://doi.org/10.1080/17576180.2025.2608756
J Lipid Res. 2025 Dec 20. pii: S0022-2275(25)00230-5. [Epub ahead of print] 100967

New Multiplex LC-MS/MS Method for lipid biomarker Analysis of Inherited Neurodegenerative Metabolic Diseases.

Anna Sidorina, Giulio Catesini, Federica Deodato, Sara Boenzi, Diego Martinelli, Cristiano Rizzo, Carlo Dionisi-Vici.

  A significant number of inherited neurodegenerative metabolic diseases (NMD) arise from altered lipid metabolism, including impaired degradation of sphingolipids and dysfunction in organelle-related machineries involved in lipid processing and trafficking. These lipid dysregulations profoundly impact cellular membranes, signaling pathways, and myelin integrity, contributing to the complex and multisystemic clinical phenotypes characteristic of NMD, which often complicate diagnosis and delay treatment initiation. Here, we present a high-throughput, multiplex liquid chromatography-mass spectrometry (LC-MS/MS) method for the analysis of an extended panel of NMD biomarkers in plasma and dried blood spots. One-step sample extraction and targeted LC-MS/MS acquisitions in positive and negative ionization allowed the simultaneous meausurement of 13 diagnostic biomarkers associated with GM1 and GM2 gangliosidosis, Fabry, Gaucher, and Krabbe diseases, acid sphingomyelinase deficiency (ASMD), Niemann-Pick disease type C (NPC), X-linked adrenoleukodystrophy (X-ALD), peroxisomal biogenesis disorders (PBD, Zellweger syndrome), metachromatic leukodystrophy (MLD), and MEDNIK/MEDNIK-like syndromes, a disorder of cellular trafficking. The method was analytically and clinically validated, confirming the diagnosis of all targeted NMDs in samples from 89 patients. Additionally, the method allowed the differentiation of X-ALD from PBD and revealed the elevation of C18- and C16-sulfatides in Krabbe-disease and MEDNIK-syndromes, respectively. This multiplex assay enhances diagnostic efficiency and expands the discovery of novel biomarkers, enabling the quantification of diagnostic markers for a wide range of NMDs. The method is suitable for diagnosis of NMD, as a first- or second-tier test in neonatal screening, as confirmatory testing of variant of unknown significance in genetic panels and for longitudinal monitoring in treatable diseases.

Keywords:  Biomarker analysis; Brain lipids; Ceramides; Cerebrosides; Glycolipids; Lysophospholipid; Neurodegenerative metabolic diseases; Newborn screening; Sphingolipids; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jlr.2025.100967
Mass Spectrom (Tokyo). 2025 ;14(1): A0181

Fragmentation Patterns of Chlorogenic Acid Homologs in Positive- and Negative-Ion Mode Mass Spectrometry.

Atsushi Yamamoto.

  Chlorogenic acids, esters of hydroxycinnamic acids with quinic acid, are abundant plant metabolites with over 400 known derivatives. Due to the limited availability of commercial standards, mass spectrometry fragmentation data are essential for structural identification. We acquired fragmentation spectra of six chlorogenic acid homologs in both positive- and negative-ion modes using direct infusion mass spectrometry. In positive-ion mode, sodiated molecules provided additional structural information in addition to that from protonated molecules, although the difference in substitution positions had minimal effects on fragmentation patterns. In negative-ion mode, fragmentation differed significantly depending on the acyl group substitution position on the quinic acid moiety, enabling isomer differentiation. This positional selectivity in negative-ion fragmentation parallels previous observations with anhydrous monosaccharides and oligosaccharides. Comparative analysis with maltotriose and β-glucan trisaccharides demonstrated that negative-ion mode fragmentation yields more diagnostic ring cleavage information for structural characterization. This study also emphasizes that the adoption of unambiguous IUPAC (International Union of Pure and Applied Chemistry)-based nomenclature is fundamental to ensuring the reliability of mass spectra databases.

Keywords:  chlorogenic acid; structural elucidation; substitution isomer discrimination

DOI:  https://doi.org/10.5702/massspectrometry.A0181
Anal Chem. 2025 Dec 24.

Spatial Ion Compression without Loss of Resolution on a Cyclic Ion Mobility Spectrometry-Mass Spectrometry Platform.

Paige E Robinson, Noah D Roberts, Gabe Nagy.

Traveling wave-based ion mobility spectrometry-mass spectrometry (IMS-MS) has emerged as an analytical technique, particularly because of technological improvements for enabling the scalable resolution of challenging biomolecules. However, IMS-MS remains limited by peak broadening from diffusion in extended separations, thus precluding further gains in sensitivity and resolution. Previous efforts have attempted to overcome these issues through the development of peak compression strategies, such as compression ratio ion mobility programming (CRIMP) and temporal compression. Unfortunately, both previous compression strategies suffer from certain drawbacks related to resolution. Herein, we present a new reinjection-based spatial ion compression strategy implemented on a commercially available cyclic IMS-MS platform without hardware or software modifications. Our method involves slicing a portion of a mobility peak, storing it in the prestore, and reinjecting it under conditions ensuring a gentle transition back to separation conditions as well optimizing timing for the remerging (i.e., spatial compression) step. Our spatial ion compression approach enables reduction in peak widths, improved sensitivity without any ion losses during reinjection, and allows continued separation after the compression event. We highlight how this compression strategy can enable better feature finding by improving peak intensity and signal-to-noise of a low abundance species as well as achieving near baseline resolution of previously partially resolved compounds. Overall, our reinjection-based spatial ion peak compression strategy can be readily adaptable on any traveling wave-based IMS-MS platforms with ion storage regions (i.e., traps) and is an added one to the suite of existing compression strategies in IMS-MS measurements.

DOI: https://doi.org/10.1021/acs.analchem.5c05855
Anal Chem. 2025 Dec 26.

Chiral Derivatization Enables High-Resolution Ion Mobility Spectrometry of 30 Amino Acid Enantiomers.

Chi Yan, Xingyu Chen, Wenqing Gao, Chunlan Tang, Liwen Du, Chengyi Xie, Feng Xu, Keqi Tang, Jiancheng Yu.

Chiral analysis of amino acid enantiomers is essential due to their frequently divergent biological activities. However, ion mobility spectrometry-mass spectrometry (IMS-MS) cannot directly resolve underivatized amino acid enantiomers due to their identical physicochemical properties. This study developed a derivatization-based analytical method utilizing (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE) as a chiral derivatization agent to achieve the stereoselective modification of amino acids. The resulting diastereomers form metal ion adducts, which enhance collision cross section differences and enable separation via trapped ion mobility spectrometry and time-of-flight mass spectrometry (TIMS-MS). The differential impact of alkali metal ion adduction (Na+, K+, Rb+, and Cs+) on chiral separation was investigated, revealing that Na+ adducts provide optimal enantioselective recognition (average resolution, Rpp = 2.02). The established method achieved separation for 30 chiral amino acids, demonstrating faster speed, broader coverage, and superior resolution compared with existing approaches. When applied to rat blood samples from chlorfenapyr-induced toxicity models, the method successfully detected d-Arg, d-Ile, d-Leu, d-Phe, and d-Met, confirming its practical utility in toxicological diagnostics and biomarker discovery.

DOI: https://doi.org/10.1021/acs.analchem.5c06382
Biomed Chromatogr. 2026 Feb;40(2): e70316

Analytical Techniques for Quantification of Metoclopramide: A Comprehensive Review.

Hemn A H Barzani, Rebaz Anwar Omer, Khalamala Ibrahim Salih Barzani, Zanco Hassan Jawhar, Seerwan Hamadameen Sulaiman.

  Metoclopramide (MCP) is a commonly used prokinetic and antiemetic medication prescribed for managing gastrointestinal motility disorders, nausea, and vomiting. The risk of adverse reactions, as well as accurate quantification of MCP in pharmaceutical products and biological samples, is essential for quality control, pharmacokinetic assessment, and therapeutic monitoring. Relevant studies for this review were gathered from major scientific databases, including Scopus, Web of Science, ScienceDirect, Google Scholar, and PubMed, restricted to publications written in English. This review provides a critical overview of analytical techniques reported over the last 25 years for MCP determination, focusing on chromatographic, spectroscopic, and electroanalytical methods. Chromatographic techniques-particularly high-performance liquid chromatography (HPLC)-remain the gold standard due to their robustness, precision, and compatibility with diverse sample matrices. Advanced LC-MS/MS systems offer exceptional sensitivity, reaching pg-ng/mL levels, which is particularly valuable in bioanalytical applications. HPLC-UV approaches, though less sensitive, are cost-effective and suitable for routine quality assessment. Spectroscopic methods such as UV-Vis spectroscopy are rapid and affordable but limited by poor selectivity in complex matrices. Electrochemical and voltammetric methods have recently gained attention, offering low cost, speed, and remarkable sensitivity, especially with nanomaterial-modified electrodes. Remaining challenges include MCP instability, matrix interferences, and balancing sensitivity with accessibility.

Keywords:  analytical methods; chromatography; electrochemical analysis; metoclopramide; spectroscopy; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/bmc.70316
Anal Methods. 2025 Dec 23.

Determination of paraquat in drinking water by HPLC-UV using large-volume injection and a superficially porous particle HILIC column.

Ismael Dos S Belmonte, Tânia M Pizzolato, Mariana R Gama.

Paraquat is a highly polar herbicide that can be detected in environmental water samples due to its unauthorized use in agriculture and its high mobility in aquatic systems. Its determination at trace levels remains analytically challenging, particularly when using conventional multiresidue methods based on reversed-phase liquid chromatography, which often requires ion-pairing reagents that compromise the method precision. In this work, the application of a large-volume injection (LVI) strategy was exploited for a superficially porous (core-shell) column to enhance the sensitivity of paraquat detection in tap water samples using HPLC with UV detection at 257 nm. The hydrophilic interaction liquid chromatography (HILIC) enabled paraquat retention, with a mobile phase composed of 50 mmol L-1 ammonium formate (pH 3,0) and acetonitrile (25 : 75, v/v). The method employed an injection volume of 50 µL and provided robust quantification of paraquat in tap water, achieving a limit of quantification of 2.5 µg L-1, excellent linearity (R2 = 0.9999), and recovery values between 98.3 and 107.0%, without matrix effects. The strategy crucially avoided SPE preconcentration and waste generation. The proposed method circumvented the sample volume limitations typically associated with core-shell columns, achieving a simple and cost-effective approach for paraquat environmental monitoring.

DOI: https://doi.org/10.1039/d5ay01675d
J Chromatogr A. 2025 Dec 16. pii: S0021-9673(25)00976-8. [Epub ahead of print]1767 466632

Screening of active ingredients from traditional Chinese medicine using two-dimensional liquid chromatography-mass spectrometry: A case study of screening neuraminidase inhibitors from Schizonepetae spica.

Hongping Wang, Zhaozhou Lin, Quantao Ma.

  The traditional methods of screening active ingredients from traditional Chinese medicine (TCM) are primarily based on chemical separation followed by activity evaluation, which involve long experimental cycles and large workloads. However, identifying active ingredients in TCM based on the targets related to a disease results in significantly improved screening efficiency. In this study, we developed a method for screening active components using two-dimensional liquid chromatography coupled with mass spectrometry (2D LC-MS). Briefly, the TCM extract was co-incubated with the target to form target-ligand complexes. Then, the incubation solution was loaded onto a 1D size-exclusion chromatography column, where the target-ligand complexes were separated from the unbound free compounds. Subsequently, a 2D reversed-phase chromatography column was used to dissociate the target-ligand complexes. Chromatographic separation was further optimized to ensure separation of the released ligands and inactive target. Finally, the dissociated ligands were analyzed using high-resolution mass spectrometry for structural identification. As an example, we screened the neuraminidase inhibitors from Schizonepetae spica using the method developed in this study and successfully identified a total of 40 ligands. The proposed 2D LC-MS method can be used for screening other active ingredients in TCM, and the screening model for neuraminidase inhibitors established in this study can be used for screening neuraminidase inhibitors from other TCM.

Keywords:  LC-MS; Neuraminidase inhibitor; Schizonepetae spica; Two-dimensional liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2025.466632
J Pharm Biomed Anal. 2025 Dec 19. pii: S0731-7085(25)00662-4. [Epub ahead of print]271 117321

Development and validation of a rapid LC-MS/MS method for methylphenidate, atomoxetine, and their metabolites with application to pediatric pharmacokinetics.

Duygu Eryavuz Onmaz, Merve Kuz, Fatih Hilmi Cetin, Halit Necmi Ucar, Serhat Turkoglu, Rabia Coban, Ali Unlu.

  Therapeutic drug monitoring of attention-deficit/hyperactivity disorder (ADHD) pharmacotherapy is challenged by substantial interindividual variability and the lack of rapid, multiplex analytical platforms. We developed and validated a fast, integrated 5-minute LC-MS/MS assay for the simultaneous quantification of stimulant (methylphenidate, ritalinic acid) and non-stimulant (atomoxetine, 4-hydroxy-atomoxetine) ADHD medications in human serum. The method employs a single-step protein precipitation and requires only 200 µL of serum. Validation demonstrated excellent linearity, precision, accuracy, minimal matrix effects, and robust analyte stability in accordance with FDA and CLSI guidelines. Application to pediatric ADHD patients revealed formulation- and dose-dependent concentration differences for methylphenidate and atomoxetine, along with strong parent-metabolite correlations. No significant associations were observed with age, sex, or body mass index. This rapid and reliable LC-MS/MS platform supports clinically actionable therapeutic drug monitoring and provides a practical framework for personalized ADHD treatment.

Keywords:  Atomoxetine; LC–MS/MS; Methylphenidate; Pediatric ADHD; Pharmacokinetics; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jpba.2025.117321
J Proteome Res. 2025 Dec 26.

Early Detection of Infectious Diseases: A Review of Recent Advances in Pathogen Identification, Molecular Tools, and Metabolomics-Driven Biomarker Discovery.

Patrica-Ivy Agorsor, Michael O Eze.

  The recent COVID-19 pandemic has heightened public interest in noninvasive methods for early diagnosis of infectious diseases. In addition, various government agencies have implemented "infectious disease preparedness" to mitigate future outbreaks. This review highlights conventional and advanced methods for infectious disease diagnosis with an emphasis on emerging mass spectrometry methods. Conventional methods for pathogen identification, such as culture-based techniques and molecular methods, have limitations with respect to sensitivity, specificity, and turnaround time. Recent advances in high-resolution mass spectrometry have revolutionized the field of infectious disease biomarker discovery. These techniques enable the comprehensive profiling of metabolites in various biological samples, identification of disease-specific biomarkers, and elucidation of complex host-pathogen interactions. While liquid chromatography-mass spectrometry has been extensively used to identify metabolic alterations in diseases, such as COVID-19, tuberculosis, pneumonia, and influenza, this often requires the use of body fluids. On the other hand, advances in gas chromatography-high resolution mass spectrometry are enabling noninvasive detection of infectious diseases by means of breath-based volatile organic compounds. These methods offer high sensitivity and specificity, enabling the detection of low-abundance biomolecules and the elucidation of complex biological pathways. This review further examines the limitations of each approach while emphasizing the essential applications of metabolomics in infectious disease diagnosis.

Keywords:  biomarker discovery; diagnostic tools; infectious diseases; mass spectrometry; metabolites; molecular methods

DOI:  https://doi.org/10.1021/acs.jproteome.5c01014