bims-metlip 2026-02-15 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–02–15
38 papers selected by
Sofia Costa, Matterworks

RPLC- and HILIC-based non-targeted metabolomics workflow for blood microsamples.
Ultrasensitive Detection of Tissue Lipids at the Femtomole Scale Using Low-Microflow Targeted Lipidomics.
Evaluation of LC/MS Methods for Hydrophilic Metabolites to Enable Integration of Human Blood Metabolome Data.
Cross ionization mode chemical similarity prediction between tandem mass spectra in metabolomics.
ChemEmbed: a deep learning framework for metabolite identification using enhanced MS/MS data and multidimensional molecular embeddings.
LipidLocator: an open source Shiny web application for spatial lipidomics.
Simultaneous determination of three phosphatidylethanol homologues, 12 drugs and metabolites in whole blood by LC-MS/MS.
Methodological Approaches for Extraction, Chromatographic and Mass Spectrometric Analysis of Lipids.
CellMate─A Deep Learning-Assisted Single-Cell Data Processing Platform.
Development of a Robust, Rapid and Reliable Tandem Mass Spectrometry Method for the Measurement of Sildenafil, Bosentan and their Major Metabolites.
What's in a name? Metabolite identification: challenges and pitfalls in untargeted metabolomics.
Development and Validation of a UHPLC-MS/MS Method to Identify Interference from Qiliqiangxin Capsule and Deslanoside in Digoxin Therapeutic Drug Monitoring: A Comparison with Immunoassay.
Magnetic bead-assisted extraction combined with ID-LC-MS/MS for simultaneous quantification of fat-soluble vitamins A, D, E, and K in human serum.
Development of a High-Throughput LC-MS/MS Method for Simultaneous Quantification of Four Therapeutic Monoclonal Antibodies in Human Serum: Application in Clinical Therapeutic Drug Monitoring.
Analyzing Plant Low-Molecular-Weight Polar Metabolites: A GC-MS Approach.
Advances in sample preparation techniques for chromatographic analysis of personal care products and their metabolites in human biological matrices: A critical review.
Development of an Accurate Double Isotopic Standard LC-MS/MS Method for Hyaluronic Acid Quantification in Biological Matrices.
Synthetic cannabinoids in hair: a review of recent findings and analytical methods by LC-MS/MS.
A validated LC-ESI-MS/MS assay for simultaneous plasma quantification of 11 antimicrobials to support personalized dosing in critically ill children.
A validated UHPLC-MS/MS method for determination of BPI-43487 and its metabolite BPI-43739 in human plasma and its application to a pharmacokinetic study in Chinese patients with advanced solid tumors.
Deep-coverage single-cell metabolomics enabled by ion mobility-resolved mass cytometry.
Online supercritical fluid derivative extraction-pressure change focusing-supercritical fluid chromatography-mass spectrometry for identification of double bond positional isomers in unsaturated lipids.
Development of a High-Throughput UHPLC-DMS-MS/MS Method for Targeted Quantitation of Pertinent Phospholipid Classes in Colon Cancer.
FLARE: Fine-grained Learning for Alignment of spectra-molecule REpresentation Enhances Metabolite Annotation.
Community Curation of Microbial Metabolites Enables Biological Insights of Metabolomics Data.
Imaging Bioactive Lipid Isomers in Acetaminophen-Induced Liver Injury using Nano-DESI Tandem Mass Spectrometry.
HPLC-MS/MS Method for Monitoring of L-Asparaginase Activity by Using Asparagine and Aspartic Acid Plasma Levels.
Development and Standardization of an Ex Vivo Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS.
Rapid Generation of Tandem Mass Spectrometry Reference Libraries Using Immediate Drop-on-Demand Liquid Handling Coupled to an Open Port Sampling Interface.
Combined Approach of Chromatographic Fractionation and Raman Spectroscopy for Metabolite Profiling of Enterobacter spp. Supernatant.
Beyond lipid homochirality: Analytical strategies and biological implications.
MS/MS Mass Spectrometry Filtering Tree for Bile Acid Isomer Annotation.
A Validated Method for the Simultaneous Measurement of Tryptophan, Kynurenine, Phenylalanine, and Tyrosine by High-Performance Liquid Chromatography-Ultraviolet/Fluorescence Detection in Human Plasma and Serum.
Simple, Sensitive and Simultaneous Determination of Free D3 and K2 Vitamins in Fortified Chicken Meat Products by LC-MS/MS with Electrospray Ionisation.
DECAF: Deconvoluted Extracted Ion Chromatogram-Based Quantification of Therapeutic Oligonucleotides.
Bioanalytical UHPLC-MS/MS Method for Quantification of Terbinafine in Ungual Delivery Studies.
A new sensitive derivatization assay of PrC-210 in plasma and tissues using liquid chromatography coupled with tandem mass spectrometry.
Current Mass Spectrometry Methods for the Analysis of 7-Ketocholesterol and Related Sterols.

Metabolomics. 2026 Feb 09. 22(2): 23

RPLC- and HILIC-based non-targeted metabolomics workflow for blood microsamples.

Pauline Couacault, Michael Witting.

   INTRODUCTION: Blood microsampling (BμS) has emerged as an alternative to invasive sampling methods, including blood and plasma sampling. Several studies have shown that BμS are suitable alternatives for analyzing endogenous metabolites and for metabolomics applications. Dried blood spots (DBS) have long been used for clinical applications, particularly for newborn screening. New quantitative BμS have emerged, including volumetric absorptive microsampling (VAMS).
OBJECTIVES: We aimed to develop an extraction protocol from BµS for non-targeted metabolomics analysis using a reversed-phase liquid chromatography/mass spectrometry (RPLC-MS) method for the mid- to non-polar metabolome and a hydrophilic interaction chromatography/mass spectrometry (HILIC-MS) method for the polar metabolome, based on existing protocols from the literature. To improve coverage, two new HILIC-MS methods have been developed.
METHODS: We used an in-house RPLC-MS method for the analysis of mid- to non-polar metabolites. Two new HILIC-MS/MS methods were developed using 73 chemical reference standards of polar metabolites from various classes. To optimize extraction, five procedures were investigated and compared to identify the most appropriate protocol for extracting metabolites from BµS for non-targeted metabolomics analysis. The final workflow was optimized on both DBS and VAMS.
RESULTS AND CONCLUSION: We developed and optimized a 15-minute HILIC-MS method that included column re-equilibration. Our experiments showed that using a 20% H2O/80% MeOH (v/v) mixture for extraction, with sample rehydration, is a good compromise for detecting many metabolite features. Our extraction and LC-MS methodology covered metabolites from many pathways, including amino acids, acylcarnitines, and bile acids.

Keywords:  Blood microsampling; DBS; HILIC-MS; Non-targeted metabolomics; RPLC-MS; VAMS

DOI:  https://doi.org/10.1007/s11306-026-02402-y
Anal Chem. 2026 Feb 09.

Ultrasensitive Detection of Tissue Lipids at the Femtomole Scale Using Low-Microflow Targeted Lipidomics.

Olivia L Murtagh, Payton E Lowrey, Ku-Lung Hsu.

Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics is a widely adopted method for profiling and quantifying changes in cellular and tissue lipids in response to biological and pharmacological perturbations. Effective chromatographic separation is critical for lipid analysis, yet conventional LC flow rates often compromise either sensitivity (analytical flow) or throughput (nanoflow). Microflow chromatography has proven to be an effective intermediate option; however, few lipidomic methods have been developed on this scale. Here, we describe an optimized low-microflow (25 μL/min) LC method coupled to a triple-quadrupole mass spectrometer for sensitive targeted lipidomic analysis. A 0.5 mm inner diameter C12 column provides a stable and reproducible separation across 13 different lipid classes with femtomolar limits of detection and quantitation. Using four short (∼5 min) optimized gradients, we achieve detection of over 500 endogenous lipid molecular species across six mouse tissues, with a median CV of 13% across 36 biologically independent samples. The C12 stationary phase increases the coverage of monoacylglycerols and diacylglycerols (DAGs), the nonpolar lipid classes that are often underrepresented in existing workflows. We applied low-microflow targeted lipidomics to discover an unexpected decrease in polyunsaturated DAGs in tissues from DAG lipase-beta knockout compared with wild-type mice.

DOI: https://doi.org/10.1021/acs.analchem.5c05829
Mass Spectrom (Tokyo). 2026 ;15(1): A0188

Evaluation of LC/MS Methods for Hydrophilic Metabolites to Enable Integration of Human Blood Metabolome Data.

Yuri Imado, Masatomo Takahashi, Yuki Soma, Shunsuke Aburaya, Kohta Nakatani, Taizo Hanai, Takeshi Bamba, Yoshihiro Izumi.

  Information on candidate biomarker metabolites identified in recent disease biomarker discovery research is expected to play a key role in the future of personalized and precision medicine. Liquid chromatography mass spectrometry (LC/MS) is a powerful method for metabolomic analysis due to its comprehensive coverage and high detection sensitivity. However, the suitability of LC/MS methods for the identification and quantification of hydrophilic metabolites remains debatable. Here, we evaluated the performance of LC/MS methods combining four types of LC [hydrophilic interaction chromatography (HILIC), ion chromatography (IC) with an anion-exchange (AEX) column (AEX-IC), reversed-phase LC (RPLC) with a pentafluorophenylpropyl (PFPP) column (PFPP-RPLC), and unified-hydrophilic interaction AEX LC (unified-HILIC/AEX)], using the same Orbitrap mass spectrometer, with the aim of integrating future human plasma metabolome data. First, we conducted a qualitative performance evaluation of four LC/MS methods, HILIC/MS, AEX-IC/MS, PFPP-RPLC/MS, and unified-HILIC/AEX/MS, by analyzing 511 hydrophilic metabolite standards and NIST Standard Reference Material (SRM) 1950 (Metabolites in Frozen Human Plasma). The evaluation focused on metabolome coverage, peak width, sensitivity, and separation performance of isomers. Next, we thoroughly evaluated the quantitative performance of the four analytical methods for 63 hydrophilic metabolites in SRM 1950 using a stable isotope-labeled internal standard (SILIS) mixture derived from 13C-labeled Escherichia coli extracts. Furthermore, we successfully estimated new concentration values for 29 metabolites without certified values in SRM 1950 using quantitative data from the four LC/MS methods. We objectively evaluated the performance of the four LC/MS methods and demonstrated that absolute quantification using SILIS is effective for integrating hydrophilic metabolite data in metabolomics.

Keywords:  Escherichia coli; human plasma; intra-laboratory comparison; metabolomics; stable isotope-labeled internal standard mixture

DOI:  https://doi.org/10.5702/massspectrometry.A0188
Nat Commun. 2026 Feb 07.

Cross ionization mode chemical similarity prediction between tandem mass spectra in metabolomics.

Niek F de Jonge, Elena Chekmeneva, Robin Schmid, David Joas, Lem-Joe Truong, Justin J J van der Hooft, Florian Huber.

Mass spectrometry is a cornerstone of untargeted metabolomics, enabling the characterization of metabolites in both positive and negative ionization modes. However, comparisons across ionization modes have remained a substantial challenge due to the distinct fragmentation patterns produced by each polarity. To overcome this barrier, we present MS2DeepScore 2.0, a machine learning-based model to predict chemical similarity between mass fragmentation spectra, which works both between different and the same ionization modes. We demonstrate the utility of MS2DeepScore 2.0 in three case studies, where MS2DeepScore enabled cross-ionization mode molecular networking, enhancing data exploration and metabolite annotation. To ensure robustness, we have implemented a quality estimation method that flags spectra with low information content or those dissimilar to the training data, thereby minimizing false predictions. Altogether, MS2DeepScore 2.0 extends our current capabilities in organizing, exploring, and annotating untargeted metabolomics profiles.

DOI: https://doi.org/10.1038/s41467-026-69083-y
Brief Bioinform. 2026 Jan 07. pii: bbag054. [Epub ahead of print]27(1):

ChemEmbed: a deep learning framework for metabolite identification using enhanced MS/MS data and multidimensional molecular embeddings.

Muhammad Faizan-Khan, Roger Giné, Josep M Badia, Maribel Pérez-Ribera, Manuel Ruiz-Botella, Alexandra Junza, Jordi Capellades, Iván Pérez-López, Shipei Xing, Abubaker Patan, Laura Brugnara, Anna Novials, Joan-Marc Servitja, Maria Vinaixa, Pieter C Dorrestein, Marta Sales-Pardo, Roger Guimerà, Oscar Yanes.

  Machine learning offers a promising path to annotating the large number of unidentified MS/MS spectra in metabolomics, addressing the limited coverage of current reference spectral libraries. However, existing methods often struggle with the high dimensionality and sparsity of MS/MS spectra and metabolite structures. ChemEmbed tackles these challenges by integrating multidimensional, continuous vector representations of chemical structures with enhanced MS/MS spectra. This enhancement is achieved by merging spectra across multiple collision energies and incorporating calculated neutral losses from 38 472 distinct compounds, providing richer input for a convolutional neural network (CNN). ChemEmbed ranks the correct candidate first in over 42% of cases and within the top five in more than 76% of cases. In external benchmarks such as CASMI 2016 and 2022, ChemEmbed outperforms SIRIUS 6, the current state-of-the-art in computational metabolomics. We applied ChemEmbed to predict structures in the Annotated Recurrent Unidentified Spectra (ARUS) dataset and confirmed 25 previously unidentified compounds. These findings demonstrate ChemEmbed's potential as a robust, scalable tool for accelerating metabolite identification in untargeted mass spectrometry workflows.

Keywords:  deep learning; mass spectrometry; metabolite identification; molecular embeddings; untargeted metabolomics

DOI:  https://doi.org/10.1093/bib/bbag054
Bioinform Adv. 2026 ;6(1): vbag012

LipidLocator: an open source Shiny web application for spatial lipidomics.

Prateek Arora, Simon Isfort, Nick Kirschke, Mojgan Masoodi, Nadia Mercader.

Motivation: Spatial lipidomics enables the study of how lipids are distributed within tissues, providing insights into tissue structure and function. However, analyzing complex mass spectrometry (MS) imaging (MSI) data remains challenging due to limited tools for high-confidence annotation, especially for integrating MSI, MS, and MS/MS pipelines.
Results: We developed LipidLocator, an open-source, interactive Shiny web application as a unified spatial lipidomics pipeline. LipidLocator integrates MSI data analysis from normalization, spatial clustering, and differential abundance analysis to MS and MS/MS-based lipid annotation. We utilized LipidLocator to analyze DESI-MSI and AP-SMALDI data from adult zebrafish sections, human renal carcinoma, and mouse whole brain sections, to demonstrate its ability to segment distinct anatomical structures and tissue sub-regions and to generate high-confidence lipid profiles using integrated MS and MS/MS annotation. LipidLocator is an end-to-end open-source spatial lipidomics pipeline, facilitating lipid imaging studies in various organisms and covering different lipid detection technologies, providing a valuable and user-friendly resource for investigating lipid metabolism.
Availability and implementation: The LipidLocator application is freely available as a Docker image on Docker Hub at pratarora/lipidlocator. Installation instructions and code are available at https://github.com/MercaderLabAnatomy/LipidLocator.

DOI: https://doi.org/10.1093/bioadv/vbag012
J Pharm Biomed Anal. 2026 Jan 09. pii: S0731-7085(26)00005-1. [Epub ahead of print]273 117337

Simultaneous determination of three phosphatidylethanol homologues, 12 drugs and metabolites in whole blood by LC-MS/MS.

Marisa H Maria, Nuno R Neng, Thomas Berg.

  The use of alcohol, legal and illicit substances poses great negative consequences on health and economy worldwide. LC-MS/MS allow simultaneous determination of multiple compounds in biological matrices. The aim of this study was to develop a LC-MS/MS method for the determination of the alcohol biomarker phosphatidylethanol (PEth) - including three homologues (PEth 16:0/18:1, PEth 16:0/18:2, PEth 18:0/18:1) - cocaine and three metabolites, and 8 other drugs in whole blood. Whole blood in K2EDTA tubes was prepared by liquid-liquid extraction using heptane/ethyl acetate/2-propanol (16:64:20, v:v:v). Chromatographic separation was achieved on an Acquity BEH C18 column (50 × 2.1 mm I.D., 1.7 µm particles). Mobile phase was 0.025 % ammonia, pH 10.7 (Solvent A) and methanol (Solvent B). The method was fully validated with isotope-labelled internal standards for 10 compounds. Inter-assay precision and accuracy were within ± 16 % for all analytes at five to seven tested concentrations. Recovery was within 42-79 % for 14 compounds and 11 % for benzoylecgonine. Matrix effects were within ± 25 % for most analytes. Internal standards compensated for matrix effects for compounds that had their own internal standards. A robust, precise, and accurate LC-MS/MS method for the determinations of three PEth homologues and 12 drugs and metabolites was, developed and validated. The method is valuable, especially for detecting polydrug use and alcohol consumption. To the best of our knowledge, this is the first LC-MS/MS method for the simultaneous determination of three PEth homologues and different drugs and metabolites.

Keywords:  Basic buffer-free mobile phase; Drugs; LC-MS/MS; Liquid-liquid extraction; Phosphatidylethanol 16:0/18:1; Whole blood

DOI:  https://doi.org/10.1016/j.jpba.2026.117337
Biomed Chromatogr. 2026 Mar;40(3): e70388

Methodological Approaches for Extraction, Chromatographic and Mass Spectrometric Analysis of Lipids.

Brian S Cummings.

  Lipids are a diverse class of molecules that mediate numerous structural and functional properties of cells, including trafficking, regulation of membrane proteins, and subcellular compartmentalization. They are the main constituents of biological membranes and mediate transmembrane signaling and structural dynamics. The chemical and structural complexity of lipids makes analysis using a single experimental approach challenging. As such, multiple techniques are used to extract, separate, detect, and quantify lipids, which depend on the specific lipid species and the model being studied. Lipidomics can advance our understanding of the biochemical mechanisms by which lipid-lipid and/or lipid-protein interactions mediate cell growth and death, as well as disease, and provide valuable information for the diagnosis and prognosis of diseases. This review provides an overview of essential methods for the examination of lipids, including extraction methods, chromatographic techniques, and approaches for mass spectrometric analysis. It presents the advantages and disadvantages of commonly used extraction approaches, separation techniques, and mass spectrometry analysis. It emphasizes the need for further standardization of protocols to allow integration of data derived from different platforms. Finally, it discusses the existing opportunities in the field, especially with regard to mass spectrometry imaging and single cell analysis.

Keywords:  chromatography; extraction; lipidomics; mass spectrometry; mass spectrometry imaging; phospholipids

DOI:  https://doi.org/10.1002/bmc.70388
Anal Chem. 2026 Feb 13.

CellMate─A Deep Learning-Assisted Single-Cell Data Processing Platform.

Felix Friedrich, Cátia Marques, Ingela Lanekoff.

Mass spectrometry-based single-cell metabolomics (SCM) reveals the inherent heterogeneity of individual cells among seemingly identical cell types. Fast-scanning and high-resolving mass analyzers provide the sensitivity and specificity required to probe minuscule amounts of biological material. However, acquiring data from hundreds of individual cells to achieve statistical power results in complex data sets. This challenge is compounded by the limited availability of specialized data analysis tools for single-cell metabolomics, as many techniques depend on the use of specialized sampling and ionization probes. This results in incompatibility with conventional metabolomics data processing tools. Here, we present CellMate, a MATLAB-based data processing platform designed for single-cell metabolomics using direct infusion techniques. CellMate comprises identification and peak alignment of detected metabolites in an intuitive graphical user interface. CellMate supports customizable quantitative, targeted, and nontargeted metabolomic workflows. The untargeted workflow is enabled by a novel deep learning-based image classification algorithm that effectively distinguishes endogenous metabolites from background species. The source code, along with a compiled installer, is available at github.com /LanekoffLab/CellMate. We believe that CellMate represents a significant advancement in the single-cell metabolomics toolbox, enabling comprehensive data extraction of precious metabolite information from single cells.

DOI: https://doi.org/10.1021/acs.analchem.5c07205
Indian J Clin Biochem. 2026 Feb;41(1): 113-119

Development of a Robust, Rapid and Reliable Tandem Mass Spectrometry Method for the Measurement of Sildenafil, Bosentan and their Major Metabolites.

Mohammad Ahmad Bik, Duygu Eryavuz Onmaz, Karam Mazin Kamil Gharab, Fadime Karaman, Sedat Abusoglu, Abdullah Sivrikaya, Ali Unlu.

  Pulmonary arterial hypertension (PAH) is a lethal, progressive disease with a complex pathogenesis. Bosentan, a dual endothelin receptor antagonist, and sildenafil, a phosphodiesterase type 5 inhibitor, are used to treat PAH. In this study, we aimed to develop a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) to measure the levels of bosentan, sildenafil, and their active metabolites in patients with PAH. We have developed an LC-MS/MS measurement procedure using a liquid-liquid extraction to measure serum drug concentrations and validated the procedure according to Clinical and Laboratory Standards Institute (CLSI) protocols. Finally, the validated method was used to measure the levels of sildenafil, bosentan, and their metabolite in pediatric PAH patients. The method was linear in the range of 0.975-1000 ng/ml and 0.76-3125 ng/ml for sildenafil and bosentan, respectively. LOQ values of sildenafil and bosentan were determined as 1.95 and 1.50 ng/ml, respectively. A method for measuring the levels of sildenafil and bosentan was developed that is rapid, robust, inexpensive, and requires a small serum volume. In addition, the validated method measured these drugs' levels and metabolites in pediatric patients with PAH. The results show that the established method can routinely monitor drug levels.

Keywords:  Bosentan, PAH, Sildenafil; Tandem mass spectrometry; Validation

DOI:  https://doi.org/10.1007/s12291-024-01215-x
Metabolomics. 2026 Feb 09. 22(2): 22

What's in a name? Metabolite identification: challenges and pitfalls in untargeted metabolomics.

Georgios Theodoridis, Oliver Fiehn, Michal Holčapek, Royston Goodacre, Daniel Raftery, Robert Plumb, Timothy M D Ebbels, Michael Witting, Helen Gika, Ian D Wilson.

   BACKGROUND: The aim of metabolic phenotyping (metabotyping) is to discover and identify metabolites (including lipids) that can be used to characterize biological samples and differentiate between different physiological states. The identification of the metabolites responsible for this differentiation is essential if mechanistic understanding is to be obtained. Confident metabolite identification arguably represents the most important outcome of untargeted metabolomics studies but currently the standards used for metabolite identification reported in many publications do not strictly follow the various published guidelines and thus these identifications lack sufficient proof.
AIM OF REVIEW: In this perspective we define problems that currently plague the field of metabolite identification using MS-based techniques, particularly LC-MS, in untargeted metabolic phenotyping. Despite considerable efforts by the community (researchers, instrument manufacturers, software, and database developers) this continues to be a contentious and error-prone step in the metabolomics workflow. The majority of publications provide only sparse data on the evidence for metabolic markers "identified" and we have observed an alarming increase in the frequency of erroneous metabolite identifications. Here, we describe the problem and provide several illustrative case studies. Our goal is to raise awareness and highlight the issue of poor metabolite identification, since it is also increasingly apparent that these errors are not always recognised during the reviewing process, such that papers with potentially erroneous metabolite identities reach publication.
KEY SCIENTIFIC CONCEPTS OF REVIEW: Poor metabolite identification potentially represents an existential threat to the credibility of untargeted "discovery" metabolomics and can pollute the literature. Here we describe the aetiology of the problem and explain how and why this issue affects the field. We argue that coordinated action is required by researchers, database managers, scientific societies and the reviewers, editors and publishers of scientific journals to both acknowledge and address this important problem.

Keywords:  Biomarkers; Lipidomics; Mass spectrometry; Metabolite annotation; Metabonomics; Research integrity

DOI:  https://doi.org/10.1007/s11306-025-02387-0
Ther Drug Monit. 2026 Feb 09.

Development and Validation of a UHPLC-MS/MS Method to Identify Interference from Qiliqiangxin Capsule and Deslanoside in Digoxin Therapeutic Drug Monitoring: A Comparison with Immunoassay.

Peile Wang, Zhan Wang, Guang Chen, Jing Yang.

   BACKGROUND: Digoxin is a cardioactive drug with a narrow therapeutic range, making therapeutic drug monitoring (TDM) essential in clinical practice. We aimed to establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of digoxin, deslanoside, and three cardiac glycosides from Qiliqiangxin capsule (periplogenin, periplocymarin, and periplocin) in human plasma, and to identify their interference in digoxin TDM by comparison with the enzyme multiplied immunoassay technique (EMIT).
METHODS: Chromatographic separation was performed on a reverse-phase column with gradient elution. Mass spectrometry detection was performed using an electrospray ionization source and multiple reaction monitoring mode. Sample pretreatment was conducted using liquid-liquid extraction.
RESULTS: The five analytes exhibited good linearity across the range of 0.05-10 ng/mL. Intra- and interbatch accuracy ranged from 90.6% to 105.1%, with imprecision below 10.6%. Application of this method to 314 TDM samples showed that digoxin concentrations measured using UHPLC-MS/MS and EMIT were consistent (r = 0.972) for patients taking digoxin alone. However, owing to cross-reactivity, EMIT overestimated digoxin concentrations in patients receiving only Qiliqiangxin capsule or deslanoside compared with UHPLC-MS/MS.
CONCLUSIONS: The developed UHPLC-MS/MS method was highly sensitive and time-efficient, making it a reliable tool for digoxin TDM and identifying interference from Qiliqiangxin capsule and deslanoside.

Keywords:  Qiliqiangxin capsule; UHPLC-MS/MS; deslanoside; digoxin; therapeutic drug monitoring

DOI:  https://doi.org/10.1097/FTD.0000000000001445
Talanta. 2026 Feb 06. pii: S0039-9140(26)00175-X. [Epub ahead of print]304 129520

Magnetic bead-assisted extraction combined with ID-LC-MS/MS for simultaneous quantification of fat-soluble vitamins A, D, E, and K in human serum.

Qingqing Pan, Jing Lin, Chunlan Tang, Tao Yu, Min Shen, Guangliang Wu, Huoyan Ji.

  This study developed and validated a high-throughput method based on automated magnetic solid-phase extraction (MSPE) with isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the simultaneous and accurate quantification of five fat-soluble vitamins (VA, 25(OH)D3, 25(OH)D2, VE, and VK1) in human serum. For the MSPE, a functionalized silica-based magnetic core-shell material was synthesized, featuring a poly (polystyrene-co-divinylbenzene-co-N-vinylpyrrolidone) coating and a covalently immobilized 2-vinylbenzofuran affinity ligand linked via a hydrophilic polyethylene glycol spacer. This design facilitated efficient enrichment and purification of the target analytes. The validated method demonstrated excellent specificity, with complete chromatographic separation, without significant matrix effects or interferences. It exhibited superior sensitivity and linearity, with a satisfactory linear response (R2 > 0.995 for all analytes) and a low limit of quantification (0.15 ng/mL-0.49 μg/mL). Recoveries were between 90.48 % and 101.95 %, while precision was satisfactory with intra-day and inter-day coefficients of variation (CV) below 7.14 % and 4.68 %, respectively. The accuracy of the method was further demonstrated by analyzing NIST standard reference material SRM 968f and SRM 972a, with a mean bias within ±7 %. Compared to traditional liquid-liquid extraction (LLE) process, the automated MSPE process efficiently extracted the target analytes from serum within 11 min through systematic optimization. Furthermore, method comparison showed a good agreement between the clinical routine LLE method and the MSPE method established. In addition, the method performed well in external quality assessment schemes, also confirming high accuracy and reliability. This robust methodology provides a powerful analytical tool for the precise monitoring of fat-soluble vitamins and nutritional status assessment in clinical practice and has the potential to promote the development of laboratory automation.

Keywords:  Automation; Fat-soluble vitamins; ID-LC-MS/MS; Magnetic solid-phase extraction; Method validation

DOI:  https://doi.org/10.1016/j.talanta.2026.129520
Biomed Chromatogr. 2026 Mar;40(3): e70350

Development of a High-Throughput LC-MS/MS Method for Simultaneous Quantification of Four Therapeutic Monoclonal Antibodies in Human Serum: Application in Clinical Therapeutic Drug Monitoring.

Yuan Yao, Guang-Yao Huang, Ting-Ting Wu, Ting-Fei Tan, Feng-Mei Hu, Chao Huang, Shan Gao, An-Ping Guo, Jun-Ping Wang.

  Monoclonal antibody (mAb) therapies have revolutionized cancer treatment, significantly improving patient outcomes. However, the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs exhibit considerable variability due to nonlinear kinetics and individual differences, highlighting the need for therapeutic drug monitoring (TDM). Therefore, this study aimed to develop and validate a reliable LC-MS/MS method for the simultaneous quantification of bevacizumab, trastuzumab, rituximab, and pertuzumab in human serum and evaluate its clinical applicability. Characteristic peptides were identified using Skyline. Serum samples underwent Protein G purification and trypsin digestion. Separation used a C18 column with 0.1% FA and acetonitrile, and detection employed multiple reaction monitoring with cadonilimab as the internal standard. The method demonstrated excellent linearity (1-200 μg/mL), precision (CV < 8.9%), and accuracy (±9.8%). With a runtime of 12 min, the validated method requires only 10 μL of serum per sample and meets international validation standards, supporting the clinical monitoring of these therapies. A robust, cost-effective, and high-throughput LC-MS/MS method was successfully developed for the simultaneous quantification of four therapeutic mAbs. The method significantly reduces sample volume and analysis time while maintaining high accuracy and reproducibility, making it well-suited for routine TDM and broader clinical applications.

Keywords:  bevacizumab; liquid chromatography–tandem mass spectrometry; pertuzumab; rituximab; trastuzumab

DOI:  https://doi.org/10.1002/bmc.70350
Plants (Basel). 2026 Jan 31. pii: 445. [Epub ahead of print]15(3):

Analyzing Plant Low-Molecular-Weight Polar Metabolites: A GC-MS Approach.

Tatiana Bilova, Nadezhda Frolova, Anastasia Orlova, Svetlana Silinskaia, Akif Mailov, Veronika Popova, Andrej Frolov.

  Decades ago, the introduction of GC-MS marked a significant advancement in primary plant metabolite studies. Here, in our review, we will delve into critical aspects of the workflow, spanning the selection of an analytical platform, sample preparation, analytical acquisition, and data processing and interpretation. The exceptional separation capabilities of GC, characterized by remarkable chromatographic resolution, render it ideal for analysis of the complex plant metabolome, including the separation of isomeric compounds. The diversity of analytical platforms allows the investigation of plant metabolomes using targeted and non-targeted approaches. GC-MS, equipped with efficient extraction methods and reliable derivatization protocols for semi- and non-volatile compounds, enables qualitative and quantitative analysis of these molecules. The stability of derivatives forms the foundation for the robustness and reproducibility of GC-MS methods, and their mass spectra provide characteristic fragments for confident identification and sensitive quantification of individual metabolites. There has been key progress in the advancement of GC-MS approaches to studying plant metabolism. However, the presence of artifacts during GC-MS analysis, particularly during derivatization, is a challenge that requires careful validations, which frequently necessitate additional investigations. The feasible solutions that were achieved to overcome the limitations in GC-MS-based studies are a particular focus of the present discussion.

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

DOI:  https://doi.org/10.3390/plants15030445
J Chromatogr A. 2026 Feb 01. pii: S0021-9673(26)00093-2. [Epub ahead of print]1772 466763

Advances in sample preparation techniques for chromatographic analysis of personal care products and their metabolites in human biological matrices: A critical review.

José de Jesús Olmos-Espejel, Jerónimo Cabrera-Peralta, Araceli Peña-Alvarez, Iran Ocaña-Rios.

  The extensive use of personal care products (PCPs) - including preservatives, UV filters, insect repellents, antimicrobials, and synthetic musks - has led to widespread human exposure, making them contaminants of emerging concern. They are ingredients of products that are used daily for hygiene and skin care. While biomonitoring studies confirm the presence of both parent PCPs and their metabolites in human tissues and fluids, a critical gap exists in the review of analytical methods for analyzing them simultaneously in complex biological matrices. This review critically examines state-of-the-art analytical techniques published from 2013 to the present for the simultaneous determination of multi-class PCPs and their metabolites in human biological samples, such as urine, blood, plasma, serum, saliva, breast milk, nails, and hair. It highlights advances in sample preparation, including innovative extraction and clean-up strategies, as well as chromatographic methods coupled with mass spectrometry. Special emphasis is placed on evaluating key analytical validation parameters, including accuracy, precision, limits of detection, and limits of quantification. Emerging trends reveal that parabens and their metabolites are the most frequently studied compounds, while synthetic musks remain notably underrepresented. Among biological matrices, urine and nails dominate as the preferred liquid and solid samples, respectively. To our knowledge, this is the first review to synthesize recent advances in the simultaneous analysis of diverse PCP classes and their metabolites in human matrices, offering a valuable resource for toxicologists and analytical chemists engaged in exposure assessment and biomonitoring.

Keywords:  Biological samples; Gas chromatography; Liquid chromatography; Mass spectrometry; Metabolites; Personal care products; Sample preparation techniques

DOI:  https://doi.org/10.1016/j.chroma.2026.466763
Anal Chem. 2026 Feb 11.

Development of an Accurate Double Isotopic Standard LC-MS/MS Method for Hyaluronic Acid Quantification in Biological Matrices.

Simone Manzi, Alessandra Altomare, Giacomo Mosconi, Maria Serena Rossitto, Luciano Messina, Anna Gallo, Marina Carini, Giancarlo Aldini, Giovanna Baron.

Hyaluronic acid (HA) plays key roles in tissue hydration, repair, and cellular signaling. Its quantification in biological matrices is crucial but challenging due to its endogenous nature and poor mass spectrometric detectability. We developed a robust method based on enzymatic hydrolysis, dual 13C-labeled internal standards, and the standard addition method combined with LC-MS/MS analysis. Samples from bovine vitreous humor and human synovial fluid were depolymerized with recombinant hyaluronidase to generate Δ4-mer oligomers, quantified using 100%- and 50%-13C-labeled HA as internal standards to correct the variabilities of the enzymatic digestion and MS detector. The standard addition method was used to control the matrix effects. The method showed excellent linearity (r2 > 0.99), low estimated LOD (0.147 ± 0.007 μg/mL for bovine vitreous humor and 0.143 ± 0.028 μg/mL for human synovial fluid) and LOQ (0.491 ± 0.022 μg/mL for bovine vitreous humor and 0.475 ± 0.093 μg/mL for human synovial fluid) values, high recovery (>90%), and suitable accuracy. Significant matrix effects were detected, reinforcing the need for the standard addition method. HA concentrations measured were consistent with physiological ranges. This validated strategy offers a reliable tool for HA quantification in complex biological samples, supporting both clinical and pharmaceutical applications.

DOI: https://doi.org/10.1021/acs.analchem.5c06285
Anal Chim Acta. 2026 Mar 15. pii: S0003-2670(26)00028-0. [Epub ahead of print]1391 345078

Synthetic cannabinoids in hair: a review of recent findings and analytical methods by LC-MS/MS.

Rafaela S Pereira, Olívia B O Moreira, Marcone Augusto L de Oliveira, Claudiane C Canuto, Diogo O C Lemos, Lucas M Duarte, Silvana V Rodrigues.

   BACKGROUND: Synthetic cannabinoids (SCs) represent a prominent class of new psychoactive substances (NPS), defined as substances of abuse that pose a significant public health threat. SCs are characterized by their structural diversity, high potency, and associated health risks. Their continuous emergence on the illicit drug market - driven by ongoing structural modifications - poses significant challenges for analytical detection, regulation, and toxicological evaluation. Hair analysis has proven to be a valuable biological matrix in forensic toxicology, offering an extended detection window and the ability to reflect chronic exposure.
RESULTS: This critical review focused on the application of liquid chromatography coupled to mass spectrometry (LC-MS/MS) for the determination of SCs in hair. The most important steps in hair sample preparation - collection, segmentation, decontamination, pulverization, and extraction - are evaluated in depth. The advantages and limitations of both low-resolution and high-resolution mass spectrometry are discussed. Comprehensive tables summarize the main methodological parameters related to sample preparation and instrumental conditions.
SIGNIFICANCE: Covering studies published between 2013 and 2025, this review presents an in-depth assessment of LC-MS/MS methodologies for SCs determination in hair, from sample preparation to instrumental optimization. It also provided an updated overview of SCs, considering nomenclature and toxicological effects. The findings underscore the pressing need for continued methodological advancements to improve SCs determination, supporting more accurate toxicological interpretations and providing new insights for forensic field.

Keywords:  Analytical methods; Chromatography; Forensic hair analysis; New psychoactive substances; Sample preparation

DOI:  https://doi.org/10.1016/j.aca.2026.345078
Front Mol Biosci. 2026 ;13 1745458

A validated LC-ESI-MS/MS assay for simultaneous plasma quantification of 11 antimicrobials to support personalized dosing in critically ill children.

Yuan-Yuan Zhang, Jie Wang, Hong-Li Guo, Shi-Yu Wu, Hai-Feng Zhang, Peng-Fei Zheng, Hong-Jun Miao, Feng Chen.

   Introduction: Critically ill patients with severe infections demonstrate profound alterations in pharmacokinetic behavior. Therapeutic drug monitoring (TDM)-informed antimicrobial dose optimization is thus essential in intensive care settings to ensure maximal bactericidal activity while mitigating toxicity, creating an urgent need for accessible analytical methodologies. Although various studies exist in this domain, the inherent technical constraints of current methodologies persistently prevent the full resolution of these unmet clinical requirements.
Methods: A rapid and sensitive LC-ESI-MS/MS method was developed and validated for the simultaneous quantification of 11 antimicrobials in human plasma. Sample preparation was performed by a simple one-step protein precipitation using methanol containing 0.1% formic acid. The analytes were separated on a Kinetex C18 column with a "corner-folded cleaver-shaped" gradient elution program and detected using multiple reaction monitoring (MRM) in positive ionization mode.
Results and Discussion: The gradient elution program achieved baseline separation of all tested analytes within 8 min, with symmetrical peak shapes and no endogenous interference. The developed method was proven to be free of matrix effects, excellent linearity (R 2 > 0.99 for all analytes), and met international bioanalytical validation criteria across clinically relevant concentration ranges. Notably, this validated method was successfully applied to children receiving mono- or combination therapy for infections. The assay meets requirements for clinical TDM implementation, providing pediatricians with reliable antibiotic concentration measurements within a clinically relevant timeframe.

Keywords:  LC-ESI-MS/MS; antimicrobial; children; infection; therapeutic drug monitoring

DOI:  https://doi.org/10.3389/fmolb.2026.1745458
Bioanalysis. 2026 Feb 10. 1-8

A validated UHPLC-MS/MS method for determination of BPI-43487 and its metabolite BPI-43739 in human plasma and its application to a pharmacokinetic study in Chinese patients with advanced solid tumors.

Yunhai Bo, Jing Lu, Min Jiang, Xiaohong Liu, Le Le, Xiaoyun Liu, Hong Lan, Jifang Gong, Fen Yang.

   BACKGROUND: BPI-43487 is a novel irreversible fibroblast growth factor receptor 4 inhibitor.
RESEARCH DESIGN AND METHODS: A simple and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of BPI-43487 and its active metabolite BPI-43739 in human plasma. BPI-43487B was used as the internal standards (IS). Plasma samples were protein precipitated by acetonitrile and processed samples were chromatographed on an AQUITY UHPLC BEH C18 column (50 × 2.1 mm, i.d. 1.7 μm) with acetonitrile and 10 mM ammonium acetate containing 0.1% formic acid (v/v) as the mobile phase.
RESULTS: Calibration curves demonstrated good linearity (R ≥0.99) over the concentration range of 10.0-5000 ng/mL for both analytes. Intra- and inter-batch precisions were ≤9.2% for BPI-43487 and ≤8.7% for BPI-43739. The accuracies were 94.6-104.8% for BPI-43487 and 95.0-108.4% for BPI-43739.
CONCLUSIONS: This method was further successfully applied to a pharmacokinetic study of BPI-43487 capsules in Chinese patients with advanced solid tumors.
CHINA DRUG TRIALS: http://www.chinadrugtrials.org.cn is CTR20210565).

Keywords:  BPI-43487; BPI-43739; LC-MS/MS; hepatocellular carcinoma; pharmacokinetics

DOI:  https://doi.org/10.1080/17576180.2026.2628084
Nat Methods. 2026 Feb 09.

Deep-coverage single-cell metabolomics enabled by ion mobility-resolved mass cytometry.

Mingdu Luo, Tianzhang Kou, Yandong Yin, Shengyi Zhou, Xiaolan Zhu, Xinhao Zeng, Junhao Hu, Zheng-Jiang Zhu.

Current single-cell metabolomics approaches are limited by insufficient sensitivity, robustness and metabolite coverage. We present an ion mobility-resolved mass cytometry technology that integrates high-throughput single-cell injection with ion mobility-mass spectrometry for multidimensional metabolomic profiling. Ion mobility-enabled selective ion accumulation and cell superposition-based amplification strategies substantially enhance sensitivity, robustness and overall analytical performance. Combined with our computational tool, MetCell, this technology allows high-throughput analysis while achieving exceptional profiling depth, detecting over 5,000 metabolic peaks and annotating approximately 800 metabolites per cell-representing a 3-fold to 10-fold improvement over existing methods. It offers attomole-level sensitivity and captures a broad dynamic range of metabolites within individual cells. Applied to 45,603 primary liver cells from aging mice, it enabled accurate cell-type and cell-subtype annotation and revealed distinct metabolic states and heterogeneity in hepatocytes during aging. This platform sets a new benchmark for high-throughput single-cell metabolomics, advancing our understanding of metabolic heterogeneity at single-cell resolution.

DOI: https://doi.org/10.1038/s41592-025-02970-2
Talanta. 2026 Feb 01. pii: S0039-9140(26)00150-5. [Epub ahead of print]303 129495

Online supercritical fluid derivative extraction-pressure change focusing-supercritical fluid chromatography-mass spectrometry for identification of double bond positional isomers in unsaturated lipids.

Longhui Huang, Zaixin Wu, Jiaqi Liu, Dianbao Yu, Ting Zhou.

  Precise localization of carbon-carbon double bonds in unsaturated lipids is essential for elucidating lipid functions and disease mechanisms, yet conventional liquid chromatography-mass spectrometry workflows are hampered by labor-intensive pretreatment, solvent consumption, and insufficient sensitivity. Here, we presented an online supercritical fluid derivative extraction-pressure change focusing-supercritical fluid chromatography-mass spectrometry platform that integrated derivatization, extraction, purification, separation, and detection into a single automated workflow. The supercritical fluid derivative extraction strategy enabled simultaneous in situ epoxidation and cleanup, while the pressure change focusing strategy effectively mitigated chromatographic band broadening, yielding sharper peaks and enhanced sensitivity. Systematic optimization established robust operating conditions, enabling comprehensive lipid analysis to be accomplished within 24 min using only 2.5 μL of sample. The validated method achieved excellent linearity, trueness, and recovery, showing coefficients of determination (R2) > 0.9930, recoveries of 73.8-111.8%, and trueness of 82.1-116.4% with precision better than 13.7% (RSD). The method was applied to plasma samples from schizophrenia mouse models. A total of 56 unsaturated fatty acids were identified with fully resolved positions of carbon-carbon double bonds, of which eight species exhibited significant abundance changes. Moreover, isomer ratio analysis revealed disease-associated remodeling of desaturation patterns, providing new insights into lipid metabolic dysregulation in schizophrenia. Overall, the established online platform represents a rapid, sensitive, and environmentally friendly strategy for structural lipidomics, offering strong potential for biomarker discovery and broader applications in biomedical and clinical research.

Keywords:  Pressure change focusing; Schizophrenia; Structural lipidomics; Supercritical fluid chromatography; Supercritical fluid derivative extraction

DOI:  https://doi.org/10.1016/j.talanta.2026.129495
Molecules. 2026 Jan 27. pii: 438. [Epub ahead of print]31(3):

Development of a High-Throughput UHPLC-DMS-MS/MS Method for Targeted Quantitation of Pertinent Phospholipid Classes in Colon Cancer.

Miriam Wimmer, Olivia I Coleman, Adam Sorbie, Dirk Haller, Veronika Somoza, Andreas Dunkel.

  Phospholipids are essential membrane constituents that regulate diverse cellular processes, yet most current workflows rely on relative quantification using high-resolution LC-MS. We developed and validated a highly selective targeted method that couples liquid chromatography with differential mobility spectrometry and tandem mass spectrometry (LC-DMS-MS/MS), providing enhanced selectivity and reduced background noise. The assay quantifies 63 phospholipid species across four classes, achieving excellent recoveries and limits of quantification in the low ng per mg tissue range. Applied to tissues from a colon cancer study in mice, the method enabled the absolute quantification of 47 species, 22 of which were significantly increased in tumor tissue versus adjacent non-tumor tissue. While phosphatidylcholines were the most abundant class overall, the largest fold changes were observed in long-chain phosphatidylglycerol and phosphatidylethanolamine species. LC-DMS-MS/MS thus offers a robust, selective platform for absolute phospholipid quantification and for detecting disease-associated lipid remodeling.

Keywords:  LC-DMS-MS/MS; colon cancer; phospholipid; rapid work-up

DOI:  https://doi.org/10.3390/molecules31030438
bioRxiv. 2026 Jan 28. pii: 2026.01.27.702086. [Epub ahead of print]

FLARE: Fine-grained Learning for Alignment of spectra-molecule REpresentation Enhances Metabolite Annotation.

Yan Zhou Chen, Blake Rushing, Soha Hassoun.

Accurate metabolite annotation via tandem mass spectrometry remains a major bottleneck in untargeted metabolomics. Recent implicit models that avoid molecular generation or spectra simulation have shown competitive performance by aligning spectra and molecular structures in the embedding space. Still, they overlook the detailed relationships between spectral peaks and molecular substructures that govern fragmentation. We introduce FLARE (Fine-grained Learning for Alignment of spectra-molecule REpresentations), a contrastive learning framework that leverages bidirectional peak-node alignment under learned weak supervision. Unlike models that rely solely on global embeddings, FLARE computes similarity via maxima over peak-to-atom and atom-to-peak interactions, capturing chemically meaningful local correspondences and enabling interpretable spectra-molecule matching. It achieves state-of-the-art results on MassSpecGym, with 43.15% rank@1 (mass-based) and 22.66% (formula-based), surpassing previous models by over 63%. FLARE's learned embeddings correspond with molecular classes, match fingerprint similarity, and detect differential metabolites in a breast cancer xenograft study, showcasing its translational potential.

DOI: https://doi.org/10.64898/2026.01.27.702086
bioRxiv. 2026 Jan 26. pii: 2026.01.24.701521. [Epub ahead of print]

Community Curation of Microbial Metabolites Enables Biological Insights of Metabolomics Data.

Helena Mannochio-Russo, Wilhan D Goncalves Nunes, Shipei Xing, Fernanda de Oliveira, Andres Mauricio Caraballo-Rodriguez, Paulo Wender Portal Gomes, Vincent Charron-Lamoureux, Julius Agongo, Nicole E Avalon, Tammy Bui, Lucia Cancelada, Marc G Chevrette, Andres Cumsille, Moyses B de Araujo, Marilyn De Graeve, Victoria Deleray, Mohamed S Donia, Mutsawashe B Dzveta, Yasin El Abiead, Ronald J Ellis, Donald Franklin, Neha Garg, Harsha Gouda, Claude Y Hamany Djande, Anastasia Hiskia, Benjamin N Ho, Chambers C Hughes, Sunghoon Hwang, Sofia Iliakopoulou, Jennifer E Iudicello, Alan K Jarmusch, Triantafyllos Kaloudis, Irina Koester, Robert Konkel, Hector H F Koolen, Kine Eide Kvitne, Sabina Leanti La Rosa, Anny Lam, Santosh Lamichhane, Motseoa Lephatsi, Scott Letendre, Sarolt Magyari, Hanna Mazur-Marzec, Daniel McDonald, Ipsita Mohanty, Monica Monge-Loria, David J Moore, Thiago Andre Moura Veiga, Musiwalo S Mulaudzi, Lerato Nephali, Griffith Nguyen, Martin Orsagh, Abubaker Patan, Tomas Pluskal, Phillip B Pope, Livia Soman de Medeiros, Paolo Stincone, Andrej Tekel, Sydney Thomas, Ralph R Torres, Shirley M Tsunoda, Fidele Tugizimana, Martijn van Faassen, Felipe Vasquez-Castro, Giovanni A Vitale, Berenike C Wagner, Crystal X Wang, Sevasti-Kiriaki Zervou, Haoqi Nina Zhao, Simone Zuffa, Daniel Petras, Laura-Isobel McCall, Rob Knight, Mingxun Wang, Pieter C Dorrestein.

Microbial metabolites play a critical role in regulating ecosystems, including the human body and its microbiota. However, understanding the physiologically relevant role of these molecules, especially through liquid chromatography tandem mass spectrometry (LC-MS/MS)-based untargeted metabolomics, poses significant challenges and often requires manual parsing of a large amount of literature, databases, and webpages. To address this gap, we established the Collaborative Microbial Metabolite Center knowledgebase (CMMC-KB), a platform that fosters collaborative efforts within the scientific community to curate knowledge about microbial metabolites. The CMMC-KB aims to collect comprehensive information about microbial molecules originating from microbial biosynthesis, drug metabolism, exposure-related molecules, food, host-derived molecules, and, whenever available, their known activities. Molecules from other sources, including host-produced, dietary, and pharmaceutical compounds, are also included. By enabling direct integration of this knowledgebase with downstream analytical tools, including molecular networking, we can deepen insights into microbiota and their metabolites, ultimately advancing our understanding of microbial ecosystems.

DOI: https://doi.org/10.64898/2026.01.24.701521
J Lipid Res. 2026 Feb 10. pii: S0022-2275(26)00021-0. [Epub ahead of print] 100995

Imaging Bioactive Lipid Isomers in Acetaminophen-Induced Liver Injury using Nano-DESI Tandem Mass Spectrometry.

Miranda R Weigand, Jepthe Yao Akakpo, Emerson Hernly, Syeda Nazifa Wali, Aiming Zheng, Anup Ramachandran, Hartmut Jaeschke, Julia Laskin.

  Acetaminophen (APAP) overdose is a leading cause of acute liver failure, resulting from the production of a reactive metabolite that induces hepatocyte necrosis. Current clinical treatments for APAP overdose offer limited therapeutic efficacy, highlighting the need for alternative strategies. 4-methylpyrazole (4-MP, Fomepizole) has emerged as a potential intervention to mitigate APAP toxicity in both mouse models and humans. Bioactive lipids, including eicosanoids and specialized pro-resolving mediators (SPMs) play essential roles in the inflammatory and resolution phases of APAP-induced liver injury. However, the impact of APAP overdose and 4-MP intervention on their distribution in liver tissue is poorly understood. Their low abundance and structural isomerism present challenges for mass spectrometry imaging (MSI). In this study, we use nanospray desorption electrospray ionization MSI in tandem mass spectrometry mode (nano-DESI MSI-MS/MS) for the spatial mapping of eicosanoids and SPMs in liver tissues of mice subjected to moderate APAP overdose with and without 4-MP treatment. Using NH4F as a solvent dopant, known to enhance analyte signals and leveraging MS/MS mode for isomer-specific analysis, we effectively detected low-abundance isomeric bioactive species. Our results reveal the localization of eicosanoids and SPMs in centrilobular hepatocytes following APAP overdose, correlating with APAP metabolism and hepatocyte necrosis. Notably, 4-MP treatment restores the spatial distributions of these lipids, supporting its therapeutic potential in modulating lipid-mediated inflammatory processes in APAP overdose. This study provides new insights into the lipid localization in APAP-induced liver injury and highlights the power of nano-DESI MSI for investigating lipid-driven pathology.

Keywords:  SPMs; acetaminophen; eicosanoids; isomers; nano-DESI; tandem mass spectrometry imaging

DOI:  https://doi.org/10.1016/j.jlr.2026.100995
J Mass Spectrom. 2026 Mar;61(3): e70036

HPLC-MS/MS Method for Monitoring of L-Asparaginase Activity by Using Asparagine and Aspartic Acid Plasma Levels.

Cem Kaplan, İbrahim Daniş, Durişehvar Özer Ünal.

  The effectiveness of L-asparaginase and therefore the effectiveness of acute lymphoblastic leukemia treatment will be understood by determining the substrates of the enzyme, L-asparagine and L-glutamine. For this purpose, the high-performance liquid chromatography-tandem mass spectrometry method was developed and validated by analyzing the L-asparaginase substrates asparagine and glutamine and its products aspartic acid and glutamic acid from plasma. Acetonitrile and ammonium acetate were used at 0.4 mL/min in gradient mobile phase flow using a HILIC column for chromatographic separations. The linear amino acid range was found to be 500-5000 ng/mL for asparagine, aspartic acid, and glutamic acid, and 5-50 μg/mL for glutamine, respectively. Detection limit and quantitation limit were found to be 100-500 ng/mL for asparagine, aspartic acid, and glutamic acid, and 1-5 μg/mL for glutamine, respectively. The validated method has been successfully applied to plasma samples. The method was found to be selective and reproducible.

Keywords:  L‐asparaginase activity; acute lymphoblastic leukemia; amino acid analysis; mass spectrometry; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/jms.70036
J Vis Exp. 2026 Jan 23.

Development and Standardization of an Ex Vivo Micromethod for Intracellular Quantification of Vincristine in Primary ALL Cells by LC-MS/MS.

Adrielli Caroline Soares, Guilherme Reis de-Oliveira, Samara de Sousa Mariano, Juliana Ronchi Corrêa, Amilcar Cardoso de Azevedo, Fábio Cesar Gozzo, José Andrés Yunes.

Quantifying intracellular vincristine (VCR) in primary pediatric acute lymphoblastic leukemia (ALL) cells is essential for understanding sample-specific differences in drug uptake and for supporting experimental studies using scarce patient material. Here, we present a streamlined LC-MS/MS micromethod specifically optimized for primary ALL cells obtained through patient-derived xenografts (PDX), addressing key challenges such as limited cell availability, small cell size, and the need for reproducible recovery after multiple washing steps. Time-course assays ranging from 1 to 5 h demonstrated that a 3 h incubation period yields the highest and most consistent intracellular VCR levels, with lower variability across replicates. Method optimization also established that using 2.5 × 106 cells per condition improves analytical robustness compared with 1 × 106 cells, which showed greater dispersion in repeated experiments. Additional critical refinements included the use of cold methanol to enhance protein precipitation efficiency and the incorporation of an internal standard to monitor and minimize procedural variation. The method enables sensitive and precise quantification of intracellular VCR across relevant concentration ranges, although the lowest tested dose (0.01 µM) was not quantifiable after the 3 h incubation. Overall, this micromethod provides a practical, reproducible, and scalable approach for measuring intracellular VCR in primary ALL samples. Its design supports comparative analyses, validation of ex vivo models, and future methodological applications aimed at integrating intracellular drug quantification into broader pharmacological assessments.

DOI: https://doi.org/10.3791/69535
J Am Soc Mass Spectrom. 2026 Feb 10.

Rapid Generation of Tandem Mass Spectrometry Reference Libraries Using Immediate Drop-on-Demand Liquid Handling Coupled to an Open Port Sampling Interface.

Emily A Kurfman, Vilmos Kertesz, John F Cahill.

  In metabolomics, tandem MS (MS2) fragmentation libraries are important for the identification of unknown features, but generating these libraries takes many valuable hours of instrument and operator time. Here, an immediate droplet-on-demand/open port sampling interface was used to rapidly acquire tandem MS of standards arrayed in a 96-well plate format. A workflow was developed for automated, high-throughput control of MS2 library generation. Pure standard mass spectral libraries were collected on Orbitrap and Q-TOF mass spectrometers for 192 compounds using 6 different collision energies with a throughput of 4 and 7.8 s/spectrum, respectively. Libraries were acquired using different solvent additives, precursor adducts, and ion polarities.

Keywords:  MS2; libraries; metabolomics; tandem MS; throughput

DOI:  https://doi.org/10.1021/jasms.5c00401
Int J Mol Sci. 2026 Feb 05. pii: 1564. [Epub ahead of print]27(3):

Combined Approach of Chromatographic Fractionation and Raman Spectroscopy for Metabolite Profiling of Enterobacter spp. Supernatant.

Elizaveta Denisova, Anastasia Avdyusheva, Vera Vasilieva, Elizaveta Tyshchuk, Polina Grebenkina, Andrey Korenevsky, Ivan Chelibanov, Vladimir Chelibanov, Areg Totolian, Lyudmila Kraeva, Dmitry Sokolov.

  The secretome of ESKAPE pathogens contains numerous bioactive molecules that play a key role in pathogenesis and the formation of an immunosuppressive microenvironment. However, analyzing this complex chemical composition presents significant methodological challenges. In this study, we propose a combined approach integrating chromatographic fractionation of cell-free supernatants with Raman spectroscopy to deconstruct the secretome of the clinically relevant Gram-negative pathogen-Enterobacter spp. Chromatographic separation of the Enterobacter spp. supernatant into seven fractions reduced spectral congestion and enabled identification of fraction 3 as having a unique metabolite profile, enriched in peptides (including tryptophan- and tyrosine-containing structures), nucleic acids, polysaccharides, and putative glutathione-like compounds. Notably, fraction 3 lacked markers of phenylalanine and sterol-like lipids, highlighting its distinct composition. Compared to conventional mass spectrometry and nuclear magnetic resonance, our hybrid strategy offers minimal sample preparation, preserves sample integrity for repeated analysis, avoids ionization bias, and is fully compatible with aqueous biological matrices-critical advantages for profiling labile or low-abundance metabolites in native secretomes. These findings demonstrate that the combination of preparative chromatography and Raman spectroscopy effectively resolves complex bacterial secretomes and identifies fractions potentially carrying key virulence or signaling functions.

Keywords:  ESKAPE pathogens; Raman spectroscopy; bacterial secretome; chromatographic fractionation; virulence metabolites

DOI:  https://doi.org/10.3390/ijms27031564
Talanta. 2026 Jan 27. pii: S0039-9140(26)00119-0. [Epub ahead of print]303 129464

Beyond lipid homochirality: Analytical strategies and biological implications.

Vijaya Shukla, Tatiana A Guaraca-Medina, Alessia Lodi, Stefano Tiziani.

  Nucleotides, carbohydrates, amino acids, and lipids have long been considered homochiral within mammalian systems. However, an increasing number of studies have reported a variety of chiral metabolites across various living organisms, some biologically active and others identified as potential disease biomarkers. Enantiomers of the same compound may have distinct biological activities, chemical reactivities, and metabolism, highlighting the increasing attention to molecular chirality in biomedical research. Like peptides, amino acids, and organic acids, lipids also possess chirality and are essential components of biological membranes, influencing both structure and functionality. Studies using simple model systems, like liposomes and vesicles, challenge the assumption that only homochiral membranes are stable, demonstrating comparable stability in racemic heterochiral membranes. Nevertheless, chirality within eukaryotic cells remains largely overlooked, resulting in limited understanding of its impact on lipid membrane organization, lipid-lipid and lipid-protein interactions, and the overall lipid metabolism. This gap primarily reflects the lack of robust experimental methods for chiral lipidomics profiling. This review provides a comprehensive overview of analytical techniques used for the separation and analysis of chiral lipids in complex biological samples, emphasizing advances in chromatographic and mass spectrometric techniques, and their application in disease biomarker discovery. We also discuss the structural and functional impact of chirality on phospholipid membranes and highlight future directions in chiral lipidomics research.

Keywords:  Chiral derivatization; Chiral lipidomics; Chromatographic and mass spectrometric techniques; Direct separation; Heterochirality; Homochirality

DOI:  https://doi.org/10.1016/j.talanta.2026.129464
bioRxiv. 2025 Mar 09. pii: 2025.03.04.641505. [Epub ahead of print]

MS/MS Mass Spectrometry Filtering Tree for Bile Acid Isomer Annotation.

Ipsita Mohanty, Shipei Xing, Vanessa Castillo, Julius Agongo, Abubaker Patan, Yasin El Abiead, Helena Mannochio-Russo, Simone Zuffa, Jasmine Zemlin, Alexandre Tronel, Audrey Le Gouellec, Thomas Soranzo, Crystal X Wang, Jennifer E Iudicello, Mohammadsobhan S Andalibi, Ronald J Ellis, David J Moore, Donald R Franklin, Marta Sala-Climent, Monica Guma, Robin M Voigt, Rima Kaddaruh-Dauok, Dionicio Siegel, Mingxun Wang, Lee R Hagey, Pieter C Dorrestein.

Bile acids are essential steroids regulating immunity, nutrient absorption, insulin, appetite, and body temperature. Their structural diversity is vast, but due to spectral similarities, MS/MS spectral matching often fails to resolve isomers. This study introduces a proof-of-concept workflow using a mass spectrometry query language filtering tree that distinguishes isomeric bile acids in untargeted LC-MS/MS data. Its application revealed a deoxycholyl-2-aminophenol amidate linked to whole grain consumption.

DOI: https://doi.org/10.1101/2025.03.04.641505
ACS Omega. 2026 Feb 03. 11(4): 5201-5210

A Validated Method for the Simultaneous Measurement of Tryptophan, Kynurenine, Phenylalanine, and Tyrosine by High-Performance Liquid Chromatography-Ultraviolet/Fluorescence Detection in Human Plasma and Serum.

Lucia Parráková, Cornelia A Karg, Stefanie Hofer, Pablo Monfort-Lanzas, Celina Wilgermein, Kevin Allmer, Sabine Scholl-Bürgi, Anita Siller, Harald Schennach, Simon Geisler, Dietmar Fuchs, Thomas K Felder, Johanna M Gostner.

Aromatic amino acids are precursors of neurotransmitters and immunomodulatory molecules, and their catabolism is dysregulated in various disorders associated with inflammation. This dysregulation often correlates with disease stage, symptom severity, comorbidities, quality of life, and cognitive performance, making its measurement valuable for research, diagnostics, and personalized monitoring. We developed a rapid, reliable, and cost-effective HPLC method for the simultaneous quantification of phenylalanine, tyrosine, tryptophan, and kynurenine in human serum and plasma. After protein precipitation, analytes were separated on a reversed-phase C18 column under isocratic conditions. Detection was performed based on intrinsic fluorescence for tryptophan, phenylalanine, and tyrosine and on UV absorption for kynurenine and the internal standard nitrotyrosine. The method showed linearity (R2 > 0.99) over 0.31-20 μM for kynurenine, 1.56-200 μM for phenylalanine, 0.08-200 μM for tryptophan, and 0.78-200 μM tyrosine. Limits of detection were 0.01 μM for tryptophan, 0.08 μM for tyrosine and kynurenine, and 0.39 μM for phenylalanine. Precision and accuracy were within 15%, and recovery rates ranged from 98 to 100%. Samples remained stable after processing and after three freeze-thaw cycles. Interlaboratory testing confirmed the reproducibility of the results. This validated method enables sensitive, accurate, and simultaneous quantification of key aromatic amino acids, providing a practical alternative to LC-MS/MS for routine diagnostics and biomarker studies.

DOI: https://doi.org/10.1021/acsomega.5c07457
Foods. 2026 Feb 05. pii: 570. [Epub ahead of print]15(3):

Simple, Sensitive and Simultaneous Determination of Free D3 and K2 Vitamins in Fortified Chicken Meat Products by LC-MS/MS with Electrospray Ionisation.

Mitja Križman.

  A rapid and simplified LC-MS method was developed for quantifying vitamins D3 (cholecalciferol) and K2 (menaquinone-4 and menaquinone-7) in high-fat chicken meat products. Sample preparation involves a two-step solvent extraction followed by centrifugation. Efficient separation was achieved on a Gemini C18 column, and electrospray in positive mode was used for detection. Method validation confirmed good performance and reproducibility. The method was successfully applied to both fortified and unfortified chicken pâté samples. Owing to its simplicity, robustness, and sensitivity, this approach provides a practical and reliable means for quantifying fat-soluble vitamins in complex animal-derived matrices and can serve as a foundation for broader applications in high-fat food products.

Keywords:  LC–MS; electrospray; fatty matrix; lipophilic vitamins; meat products; vitamin D; vitamin K

DOI:  https://doi.org/10.3390/foods15030570
Molecules. 2026 Feb 06. pii: 570. [Epub ahead of print]31(3):

DECAF: Deconvoluted Extracted Ion Chromatogram-Based Quantification of Therapeutic Oligonucleotides.

Piotr Prostko, Youzhong Liu, Michał Aleksander Ciach, Tatsiana Khamiakova, Thomas De Vijlder, Dirk Valkenborg.

  Accurate quantification in LC-MS experiments becomes challenging when analytes overlap both chromatographically and in mass spectra, as conventional extracted ion chromatogram-based methods can yield biased results by double-counting shared isotope signals. This limitation is particularly relevant for oligonucleotides, where degradation products and synthetic impurities frequently co-elute with the intended full-length product, complicating relative quantification. To address this, we developed DECAF, a straightforward and computationally efficient procedure for deconvoluting overlapping isotope patterns directly from MS1 data. The method models experimental isotope distributions as mixtures of theoretical templates across retention time, generating deconvoluted ion chromatograms whose peak areas accurately reflect the contributions of individual components. We demonstrate the utility of DECAF on two pharmaceutically relevant oligonucleotide mixture datasets, where it reliably estimated mixing proportions and enabled visualisation of component-specific elution profiles. Analysis of a typical sample required one to two minutes, underscoring the method's practical efficiency. DECAF provides a transparent and accessible alternative to existing commercial software, with promising applications in pharmaceutical analysis and quality control.

Keywords:  deconvolution; mass spectrometry; mixture decomposition; oligonucleotides; pharmaceutical development; quantification

DOI:  https://doi.org/10.3390/molecules31030570
Biomed Chromatogr. 2026 Mar;40(3): e70392

Bioanalytical UHPLC-MS/MS Method for Quantification of Terbinafine in Ungual Delivery Studies.

Benjamin Rossier, Emmanuel Varesio, Eric Allémann, Yogeshvar N Kalia.

  Terbinafine is considered the first-line treatment for dermatophyte-induced onychomycosis. This study aimed to develop a fast, selective, and sensitive UHPLC-MS/MS method for the quantification of terbinafine in porcine hooves, a cost-effective surrogate for human nails. The objectives were to (i) optimise specific chromatographic and detection settings, (ii) evaluate terbinafine extraction recovery, (iii) validate the method with regard to standardised operating procedure guidelines using terbinafine-d3 as an internal standard and (iv) investigate the method's performance through a preliminary study involving deposition of an in-house high-loading topical formulation into fractional laser-ablated porcine hooves. An extraction with MeOH:H2O (9:1) achieved ~90% recovery. Extracts were centrifuged, filtered and analysed under gradient conditions, with analyte and internal standard detected by selected reaction monitoring. The validated method demonstrated appropriate sensitivity to quantify concentration ranges between 1 and 200 ng/mL with a 7.2-min runtime and a limit of quantification of 1 ng/mL. Preliminary studies detected terbinafine from the formulation within intact and laser-ablated porcine hooves, corresponding to deposited amounts of 89.04 ± 4.02 and 217.49 ± 8.98 μg/cm2, respectively. In conclusion, the sensitivity and specificity of the method make it suitable for use in further investigations into ungual delivery enhancement strategies involving terbinafine formulations.

Keywords:  UHPLC–MS/MS; fractional laser ablation; nail; onychomycosis; terbinafine; ungual drug delivery

DOI:  https://doi.org/10.1002/bmc.70392
Bioanalysis. 2026 Feb 12. 1-7

A new sensitive derivatization assay of PrC-210 in plasma and tissues using liquid chromatography coupled with tandem mass spectrometry.

Bryan L Fahl, Cameron O Scarlett, William E Fahl.

   BACKGROUND: PrC-210 is a direct-acting reactive oxygen species (ROS) scavenger. It is active when administered orally, IV, or subcutaneously. Its profound efficacy in preclinical models makes it a candidate to prevent human ROS-induced i) ionizing radiation toxicities, ii) neurodegenerative disease, and iii) organ ischemia-reperfusion injuries.
RESEARCH DESIGN AND METHODS: A simple, highly sensitive, specific LC-MS/MS method was developed to measure the PrC-210 molecule in mouse plasma and tissue homogenates. To prevent oxidative degradation or disulfide formation of the PrC-210 thiol form, its free sulfhydryl group was protected by derivatization with N-ethylmaleimide (NEM), which produced the stable, quantifiable, PrC-210-NEM thioether conjugate. The PrC-210-NEM conjugate, and acyclovir used as an internal standard, were extracted from plasma or tissue homogenate with acetonitrile/0.1% formic acid.
RESULTS: The reconstituted dried extracts were chromatographed and then monitored using a triple quadrupole spectrometer operating in the positive ion spray ionization mode. The method was validated over the concentration range of 7.5 nM-5000 nM. Inter- and intra-assay precision and accuracy of PrC-210-NEM quantitation were better than 10%. The limit of PrC-210-NEM quantitation was 2.5 nM.
CONCLUSIONS: The method was applied to measure plasma and tissue homogenate concentrations of PrC-210 in over 126 mice administered either oral, itraperitoneal, or subcutaneous PrC-210 doses.

Keywords:  N-ethylmaleimide; ROS (reactive oxygen species); ROS-scavenger; aminothiol; neurodegenerative

DOI:  https://doi.org/10.1080/17576180.2026.2623031
J Steroid Biochem Mol Biol. 2026 Feb 10. pii: S0960-0760(26)00022-1. [Epub ahead of print] 106956

Current Mass Spectrometry Methods for the Analysis of 7-Ketocholesterol and Related Sterols.

William J Griffiths, Mohsen Ali Asgari, Yuqin Wang.

  In this article we summarise the current mass spectrometry methods used for analysis of 7-ketocholesterol and related sterols, focusing on the advantages of the different methods with emphasis on pre-analytical and analytical precautions to avoid artefactual ex vivo formation of these oxysterols.

Keywords:  Autoxidation; Cholesterol; Derivatisation; GC-MS; LC-MS; Oxysterol

DOI:  https://doi.org/10.1016/j.jsbmb.2026.106956