bims-metlip 2025-08-31 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–08–31
thirty-six papers selected by
Sofia Costa, Matterworks

Study of Metabolite Detectability in Simultaneous Profiling of Amine/Phenol and Hydroxyl Submetabolomes by Analyzing a Mixture of Two Separately Dansyl-Labeled Samples.
Chromatographic separation of dihydroxylated vitamin D3 and accurate quantification of 1α,25(OH)2D3 in human serum.
Metabolomics using anion-exchange chromatography mass spectrometry for the analysis of cells, tissues and biofluids.
An improved and economical LLE-LC/MS method for quantifying phosphatidylethanol: Identification and separation of isotopic cross-talk in clinical alcohol testing.
Nanoparticle-assisted strategies in mass spectrometry-based metabolite analysis: recent advances across analytical workflow.
Targeted mass spectrometry method for the determination of multiple gut-microbiota metabolites in human plasma.
Spatially Resolved Plant Metabolomics.
MALDI-TIMS-MS2 Imaging and Annotation of Natural Products in Fungal-Bacterial Coculture.
Assessment of Nitrosamine Impurities in Lurasidone Hydrochloride Using APCI-LC-MS/MS in MRM Mode for Regulatory Compliance and Stability Evaluation.
Optimization of Extraction Methods for NMR and LC-MS Metabolite Fingerprint Profiling of Botanical Ingredients in Food and Natural Health Products (NHPs).
Development and Validation of QuEChERS Extraction Coupled with Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry for the Detection of Nine Macrolides in Fish Products.
Increasing the Scale of the Mass Spectrometry Query Language Compendium with Explainable AI.
High-Throughput Quantification of Pesticide Residues in Complex Matrix (Chilli Powder) Using Liquid Chromatography Tandem Mass Spectrometry: Inter- and Intra-Day Validation.
QC4Metabolomics: Real-Time and Retrospective Quality Control of Metabolomics Data.
Development and validation of a green/blue UHPLC-MS/MS method for trace pharmaceutical monitoring.
Reversed-Phase (RP) and Hydrophilic Interaction (HILIC) Separation Mechanisms for the Assay of Nicotine and E-Cigarette Liquids.
Analysis of polychlorinated alkanes in food by liquid chromatography-tandem mass spectrometry.
Exploring Metal-Organic Framework-Coated Blades for Direct and High-Throughput Screening Analysis of Complex Biological Matrices.
Simultaneous Determination of Osimertinib and its Metabolites in Human Plasma for Clinical Applications Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
Spatial isotope deep tracing deciphers inter-tissue metabolic crosstalk.
Advances and challenges in sample preparation and analytical methods for water-soluble vitamins: Application in food, clinical, pharmaceutical samples.
[Validation Study on a Rapid and Low-priced Method for Determination of Chloroacetic Acids in Mineral Water Products].
Sensitive analysis of tyrosine kinase inhibitors in plasma using polypropylene fabric-solid phase extraction combined with liquid chromatography-tandem mass spectrometry.
Exploring 6-aza-2-Thiothymine as a MALDI-MSI Matrix for Spatial Lipidomics of Formalin-Fixed Paraffin-Embedded Clinical Samples.
Neuropeptide Characterization Workflow from Sampling to Data-Independent Acquisition Mass Spectrometry.
Automated single-cell analysis by single-cell side-orifice flow cytometry chain electrospray ionization mass spectrometry.
HPLC-MS/MS-based detection of Pseudomonas aeruginosa secondary metabolites and their application in metabolomics.
A confirmatory analytical method for post-harvest fungicides in perishable vegetables and fruits using liquid chromatography tandem mass spectrometry.
Lipidomic Profiling of Isolated Nuclei from Mice and Cultured Cells.
Analytical Approaches to Address Challenges in the Analysis of Cannabinoids in Vascular Matrices Using Mass Spectrometry.
An improved pretreatment method for efficient detection of antibiotic residues in aquatic products by LC-MS/MS.
MultiNMRFit: A software to fit 1D and pseudo-2D NMR spectra.
Stable isotope-labeled paper spray mass spectrometry enables high-sensitivity profiling of protease activity.
Improvement of Electrospray Ionization Response Linearity and Quantification in Dissolved Organic Matter Using Synthetic Deuterated Internal Standards.
Identification of Anatoxin-a and Related Metabolites in Exposed Mice Samples with a High-Resolution Mass Spectrometry Discovery Workflow.
Stable Nitrogen Isotope Analysis of Amino Acids by Orbitrap Mass Spectrometry: Application for Extraterrestrial Samples.

Metabolites. 2025 Jul 23. pii: 496. [Epub ahead of print]15(8):

Study of Metabolite Detectability in Simultaneous Profiling of Amine/Phenol and Hydroxyl Submetabolomes by Analyzing a Mixture of Two Separately Dansyl-Labeled Samples.

Sicheng Quan, Shuang Zhao, Liang Li.

   BACKGROUND: Liquid chromatography-mass spectrometry (LC-MS), widely used in metabolomics, is often limited by low ionization efficiency and ion suppression, which reduce overall metabolite detectability and quantification accuracy. To address these challenges, chemical isotope labeling (CIL) LC-MS has emerged as a powerful approach, offering high sensitivity, accurate quantification, and broad metabolome coverage. This method enables comprehensive profiling by targeting multiple submetabolomes. Specifically, amine-/phenol- and hydroxyl-containing metabolites are labeled using dansyl chloride under distinct reaction conditions. While this strategy provides extensive coverage, the sequential analysis of each submetabolome reduces throughput. To overcome this limitation, we propose a two-channel mixing strategy to improve analytical efficiency.
METHODS: In this approach, samples labeled separately for the amine/phenol and hydroxyl submetabolomes are combined prior to LC-MS analysis, leveraging the common use of dansyl chloride as the labeling reagent. This integration effectively doubles throughput by reducing LC-MS runtime and associated costs. The method was evaluated using human urine and serum samples, focusing on peak pair detectability and metabolite identification. A proof-of-concept study was also conducted to assess the approach's applicability in putative biomarker discovery.
RESULTS: Results demonstrate that the two-channel mixing strategy enhances throughput while maintaining analytical robustness.
CONCLUSIONS: This method is particularly suitable for large-scale studies that require rapid sample processing, where high efficiency is essential.

Keywords:  chemical isotope labeling (CIL) LC-MS; comprehensive metabolome profiling; dansyl labeling; rapid sample processing

DOI:  https://doi.org/10.3390/metabo15080496
J Pharm Biomed Anal. 2025 Aug 18. pii: S0731-7085(25)00466-2. [Epub ahead of print]266 117125

Chromatographic separation of dihydroxylated vitamin D3 and accurate quantification of 1α,25(OH)2D3 in human serum.

Koji Takahashi, Masaki Takiwaki, Rino Tsutsumi, Ryota Sakamoto, Kazuo Nagasawa, Yutaka Kuroda, Seketsu Fukuzawa.

  The most bioactive form of dihydroxylated vitamin D (VD) is 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), which is implicated in various physiological processes and disease development. Therefore, accurate quantification of 1α,25(OH)2D3 is essential for clinical diagnosis, VD-related disease management, and infection control. However, 1α,25(OH)2D3 quantification in biological samples is challenging because of its low concentration and interference from other dihydroxylated VD metabolites. In this study, we developed a novel assay for 1α,25(OH)2D3 quantification, based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with protein precipitation, solid-phase extraction (SPE), and derivatization of VD metabolites with 14-(4-dimethylaminophenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione (DAP-PA). In the derivatization process, the 6 R and 6S isomers of 1α,25(OH)2D3 obtained in a 1:1 ratio were quantitatively separated and detected using a pentafluorophenyl-octadecylsilyl mix mode column. This approach enabled the complete separation of 1α,25(OH)2D3 from other dihydroxylated VD3s, including 1β,25-dihydroxyvitamin D3 (1β,25(OH)2D3), 3-epi-1α,25-dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3), 2α,25-dihydroxyvitamin D3 (2α,25(OH)2D3), 2β,25-dihydroxyvitamin D3 (2β,25(OH)2D3), 4α,25-dihydroxyvitamin D3 (4α,25(OH)2D3), and 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3). To demonstrate the applicability of this optimized method, we analyzed human serum and compared the results with a widely used immunoaffinity extraction-based LC-MS/MS assay for 1α,25(OH)2D3, showing equivalent quantitative values. The optimized LC-MS/MS method achieved a run time of 7.7 min, with a lower limit of quantification of 2.5 pg/mL using a sample serum volume of 100 µL. This method is highly sensitive, precise, and capable of high-throughput analysis, providing a robust approach for accurate quantification of 1α,25(OH)2D3 while effectively separating interfering dihydroxylated VD3 isomers.

Keywords:  14-(4-dimethylaminophenyl)-9-phenyl-9,10-dihydro-9,10-[1,2]epitriazoloanthracene-13,15-dione (DAP-PA); 1α,25(OH)(2)D(3); 1β,25(OH)(2)D(3); 4β,25(OH)(2)D(3); Pentafluorophenyl-octadecylsilyl (PFP-C18); Vitamin D

DOI:  https://doi.org/10.1016/j.jpba.2025.117125
Nat Protoc. 2025 Aug 22.

Metabolomics using anion-exchange chromatography mass spectrometry for the analysis of cells, tissues and biofluids.

Rachel Williams, John Walsby-Tickle, Ingvild Comfort Hvinden, Isabelle Legge, Tereza Kacerova, KyoungEun Vicky Lee, Mariya Misheva, David Hauton, Judith B Ngere, John D Sidda, Elisabete Pires, Tom Cadoux-Hudson, James S O McCullagh.

The direct coupling of ion-exchange chromatography with mass spectrometry using electrochemical ion suppression creates a hyphenated technique with selectivity and specificity for the analysis of highly polar and ionic compounds. The technique has enabled new applications in environmental chemistry, food chemistry, forensics, cell biology and, more recently, metabolomics. Robust, reproducible and quantitative methods for the analysis of highly polar and ionic metabolites help meet a longstanding analytical need in metabolomics. Here, we provide step-by-step instructions for both untargeted and semi-targeted metabolite analysis from cell, tissue or biofluid samples by using anion-exchange chromatography-high-resolution tandem mass spectrometry (AEC-MS/MS). The method requires minimal sample preparation and is robust, sensitive and selective. It provides comprehensive coverage of hundreds of metabolites found in primary and secondary metabolic pathways, including glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, purine and pyrimidine metabolism, amino acid degradation and redox metabolism. An inline electrolytic ion suppressor is used to quantitatively neutralize OH- ions in the eluent stream, after chromatographic separation, enabling AEC to be directly coupled with MS. Counter ions are also removed during this process, creating a neutral pH, aqueous eluent with a simplified matrix optimal for negative ion MS analysis. Sample preparation through to data analysis and interpretation is described in the protocol, including a guide to which metabolites and metabolic pathways are suitable for analysis by using AEC-MS/MS.

DOI: https://doi.org/10.1038/s41596-025-01222-z
Clin Biochem. 2025 Aug 22. pii: S0009-9120(25)00123-7. [Epub ahead of print] 110994

An improved and economical LLE-LC/MS method for quantifying phosphatidylethanol: Identification and separation of isotopic cross-talk in clinical alcohol testing.

Naga Veera Yerra, Christopher Pini, Carlos Cordon-Cardo, Damodara Rao Mendu.

   OBJECTIVE: Phosphatidylethanol (PEth) is a group of phospholipids formed in the presence of ethanol on the red blood cell membrane. Due to their stability in blood for 3-4 weeks, they have become reliable direct biomarkers for long-term monitoring of alcohol use. This study aimed to develop and validate a robust, high-throughput liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for the routine clinical quantification of the two most common PEth homologues, PEth 16:0/18:1 (POPEth) and PEth 16:0/18:2 (PLPEth), while addressing common analytical challenges.
METHODS: An established quantification method employing liquid-liquid extraction was used with optimized LC-MS/MS parameters. The method was validated for correlation studies, precision, analytical measurement range, analytical sensitivity, analytical specificity, carryover, dilution linearity, stability, matrix effect and extraction recovery, with specific attention to eliminating isotopic cross-talk and chromatographic interferences. A method comparison was performed using specimens analyzed by an external reference laboratory.
RESULTS: The method demonstrated excellent linearity for both POPEth and PLPEth across the analytical measurement range (10-2000 ng/mL), with correlation coefficients (r2) of 0.99. Intra- and inter-assay precision values were within ± 10 % coefficient of variation. Recovery yields ranged from 78-85 %. The optimized method resolved isotopic cross-talk and exhibited no carryover. Comparison with an external laboratory showed strong correlation for both homologues (slopes of 0.979 and 1.049; r2 = 0.99).
CONCLUSION: We developed and validated a sensitive and specific LC-MS/MS method for the quantification of POPEth and PLPEth. The assay provides improved recovery, eliminates isotopic cross-talk, shows no carryover, and is suitable for high-throughput clinical laboratories. This method enables reliable and cost-effective monitoring of alcohol use in routine clinical practice.

Keywords:  Alcohol biomarker; Analytical method validation; Isotopic cross-talk; PEth 16:0/18:1; PEth 16:0/18:2

DOI:  https://doi.org/10.1016/j.clinbiochem.2025.110994
Mikrochim Acta. 2025 Aug 23. 192(9): 618

Nanoparticle-assisted strategies in mass spectrometry-based metabolite analysis: recent advances across analytical workflow.

Roberto Gamboa-Becerra, Ernesto Beltrán-Partida, Benjamín Valdez-Salas, Jhonathan Castillo-Saenz, Jorge Salvador-Carlos, Mario Curiel-Álvarez.

  Metabolite analysis plays a critical role in understanding phenotypic variations, biochemical processes, and physiological responses in biological systems. Whether through untargeted metabolomic profiling or targeted approaches aimed at quantifying specific or even individual metabolites, accurate detection presents significant analytical challenges due to their vast chemical diversity, low abundance, and complexity of biological matrices. This chemical analytical process encompasses a dynamic workflow that includes sample collection, extraction, enrichment, separation, and detection. Recent advances in nanotechnology offer promising alternatives to support and enhance each stage of this workflow, particularly within mass spectrometry (MS)-based applications. Nanoparticles, due to their high surface area, tunable surface chemistry, and ability to improve sensitivity, have been widely applied to improve sample pretreatment, selective enrichment, separation efficiency, and ionization, ultimately enhancing MS-based metabolites detection. This review provides an updated overview of nanoparticle-assisted strategies throughout the MS-based metabolite analysis workflow. It discusses the different classes of those nanomaterials and their applications across various phases stages, from sample preparation to ionization and detection, supporting analyses that range from untargeted and targeted metabolomics to the detection of individual metabolites. Although the primary focus is on MS-based workflows, we also reviewed nanoparticle-assisted separation strategies coupled with alternative detection platforms, such as optical or electrochemical methods, when these approaches show potential for integration with MS workflows. This inclusion reflects the current gap in literature addressing nanoparticle-assisted separation directly coupled with MS detection systems. These cases highlight underexplored opportunities where nanomaterials could enhance separation prior to MS detection, although further work is needed to ensure compatibility with MS platforms for suitable metabolite analysis. Furthermore, we highlight emerging trends and future perspectives in this evolving field, emphasizing the potential of nanotechnology to overcome current analytical limitations and expand the scope of both metabolomic profiling and focused metabolite analysis.

Keywords:  Analytical techniques; Analytical workflow optimization; Detection sensitivity enhancement; Nanoparticle-assisted metabolomics; Sample pretreatment; Selective metabolite enrichment; Targeted metabolite derivatization

DOI:  https://doi.org/10.1007/s00604-025-07473-7
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 14. pii: S1570-0232(25)00318-6. [Epub ahead of print]1265 124764

Targeted mass spectrometry method for the determination of multiple gut-microbiota metabolites in human plasma.

Sara R Fernandes, Cristian Azorín, Eduarda M P Silva, Manuel Miró, Luisa Barreiros, Marcela A Segundo.

  The gut microbiota profoundly impacts human health by producing metabolites that can act as biomarkers for disease diagnosis and therapy. However, accurately measuring these metabolites in biomatrices is challenging due to their low concentrations, high molecular diversity, and interference from matrix components, demanding advanced and precise analytical methodologies. Hence, an ultra-high-performance liquid chromatography method coupled to triple quadrupole-tandem mass spectrometry detection, combined with a chemical derivatization procedure, was developed and validated to quantify seven gut metabolites, namely acetic acid, propionic acid, butyric acid, p-cresol sulfate, 3-indoxyl sulfate, indole-3-acetic acid, and L-tryptophan, in human plasma. Samples were prepared by protein precipitation with acetonitrile and subsequently derivatized using 3-nitrophenylhydrazine. Chromatographic separation was achieved using a BEH C18 column, with elution performed at a flow rate of 0.2mLmin-1 and in gradient mode using formic acid-water (1:1000, v/v) and formic acid-acetonitrile (1:1000, v/v) as mobile phase components. The mass spectrometer was operated in negative ionization mode and data was acquired in selected reaction monitoring. Good linearity was achieved (r2 > 0.997) for all the target gut metabolites in the evaluated concentration ranges, with low LLOQ values (0.4-8 μM). The method proved to be accurate (87.0-114 %) and precise (CV ≤ 13.5 %), achieving a score of 65 in the Blue Applicability Grade Index (BAGI) metric, which confirmed its practicality. The developed method was ultimately employed to the analysis of plasma samples from children and adults involved in clinical studies, demonstrating its usefulness in medical research.

Keywords:  Biomatrices; Chemical derivatization; Clinical diagnostics; Gut metabolites; Mass spectrometry; Targeted analysis

DOI:  https://doi.org/10.1016/j.jchromb.2025.124764
Metabolites. 2025 Aug 08. pii: 539. [Epub ahead of print]15(8):

Spatially Resolved Plant Metabolomics.

Ronald J Myers, Zachary M Tretter, Abigail G Daffron, Eric X Fritschi, William Thives Santos, Maiya L Foster, Matthew Klotz, Kristin M Stafford, Christina Kasch, Thomas J Taylor, Lillian C Tellefson, Tyler Hartman, Dru Hackler, Preston Stephen, Lloyd W Sumner.

  Research and innovation in metabolomics tools to measure metabolite accumulation within plants have led to important discoveries with respect to the improvement of plant stress tolerance, development, and crop yield. Traditional metabolomics analyses have commonly utilized gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, but these methods are often performed without regard for the spatial locations of metabolites within tissues. Methods for mass spectral imaging (MSI) have recently been developed to detect and spatially resolve metabolite accumulation and are rapidly being adopted on a wider scale. Since 2010, the number of publications incorporating mass spectral imaging has grown from approximately 80 articles to over 378 on a yearly basis, constituting an increase of at least 350% during this time frame. Spatially resolved metabolite accumulation data provides unique insights into the function and regulation of plant biochemical pathways. Mass spectral imaging is commonly paired with desorption ionization technologies, including matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI), to generate accurate, spatially resolved metabolomics data from prepared tissue segments. Here, we describe the most recent advancements in sample preparation methods, mass spectral imaging technologies, and data processing tools that have been developed to address the limits of MSI technology. Additionally, we summarize recent applications of MSI technologies in plant metabolomics and discuss potential avenues for future research advancements within the plant biology community through the use of these technologies.

Keywords:  desorption electrospray ionization; mass spectral imaging; mass spectrometry; matrix-assisted laser desorption ionization; plant metabolism; plant stress; spatial metabolomics

DOI:  https://doi.org/10.3390/metabo15080539
Anal Chem. 2025 Aug 27.

MALDI-TIMS-MS2 Imaging and Annotation of Natural Products in Fungal-Bacterial Coculture.

Robert A Shepherd, Gordon T Luu, Laura M Sanchez.

Mass spectrometry imaging (MSI) is a powerful tool for monitoring the spatial distributions of microbial metabolites directly from culture. MSI can identify secretion and retention patterns for microbial metabolites, allowing for the assessment of chemical communication within complex microbial communities. Microbial imaging via matrix-assisted laser desorption/ionization (MALDI) MSI remains challenging due to high sample complexity and heterogeneity associated with the required sample preparation, making annotation of molecules by MS1 alone challenging. The implementation of trapped ion mobility spectrometry (TIMS) has increased the dimensionality of MALDI-MSI experiments, allowing for the resolution of isomers and isobars, and can increase sensitivity of metabolite detection within complex samples. Parallel reaction monitoring-parallel accumulation serial fragmentation (prm-PASEF) leverages TIMS to enhance the targeted acquisition of MS2 data by increasing the number of precursors that can be fragmented in a single acquisition. Recently, imaging prm-PASEF (iprm-PASEF) has been developed to provide more accurate annotation from MALDI-TIMS-MSI data sets through the inclusion of MS2. Here, we showcase the use of MALDI iprm-PASEF to provide rapid and accurate annotation of coproporphyrin III directly from a bacterial-fungal coculture between Glutamicibacter arilaitensis (strain JB182) and Penicillium solitum (strain #12). Additionally, we present a workflow for untargeted iprm-PASEF precursor selection directly in SCiLS Lab, followed by direct export for iprm-PASEF acquisition.

DOI: https://doi.org/10.1021/acs.analchem.5c02787
Rapid Commun Mass Spectrom. 2025 Dec 15. 39(23): e10126

Assessment of Nitrosamine Impurities in Lurasidone Hydrochloride Using APCI-LC-MS/MS in MRM Mode for Regulatory Compliance and Stability Evaluation.

Anant D Yadav, Ashish C Alaspure, Pinal S Bhavsar, Sandesh R Lodha, Anil H Gore.

   RATIONALE: Nitrosamine impurities, such as N-nitroso diethylamine (NDEA), N-nitroso dimethylamine (NDMA), and 1-nitroso piperazine (1-NP), have raised serious health concerns due to their carcinogenic and mutagenic properties. The pharmaceutical industry faces increasing regulatory pressure to monitor these compounds at trace levels. This study addresses the need for a robust analytical method to ensure the safety of lurasidone hydrochloride (LUR), an antipsychotic drug.
METHODS: A sensitive and specific LC-MS/MS method was developed using atmospheric pressure chemical ionization (APCI) in positive mode and operated in multiple reaction monitoring (MRM). Chromatographic separation was performed on a Sapphirus C18 HP-classic column under gradient elution, with 0.1% formic acid in water and methanol as the mobile phase. Detection was carried out using an Agilent 6470B triple-quadrupole mass spectrometer.
RESULTS: The method demonstrated excellent linearity (regression coefficient > 0.999) across a range extending to 200% of the specified limit. Limits of detection and quantitation were suitable for trace-level analysis. Accuracy was confirmed through recovery studies, with results ranging between 80 and 120%. The method met ICH Q2 (R2) validation criteria, ensuring reliability for regulatory use.
CONCLUSIONS: The developed APCI-LC-MS/MS method provides accurate and reproducible quantification of NDEA, NDMA, and 1-NP in lurasidone hydrochloride. Its sensitivity and compliance with regulatory standards make it suitable for routine quality control and stability testing of nitrosamine impurities in lurasidone hydrochloride drug substance.

Keywords:  1‐nitroso piperazine (1‐NP); ICH; N‐nitroso diethylamine (NDEA); N‐nitroso dimethylamine (NDMA); drug substance; lurasidone hydrochloride (LUR)

DOI:  https://doi.org/10.1002/rcm.10126
Molecules. 2025 Aug 14. pii: 3379. [Epub ahead of print]30(16):

Optimization of Extraction Methods for NMR and LC-MS Metabolite Fingerprint Profiling of Botanical Ingredients in Food and Natural Health Products (NHPs).

Varathan Vinayagam, Arunachalam Thirugnanasambandam, Subramanyam Ragupathy, Ragupathy Sneha, Steven G Newmaster.

  Metabolite fingerprint profiling is a robust tool for verifying suppliers of authentic botanical ingredients. While comparative studies exist, few apply identical conditions across multiple species; this study utilized a cross-species comparison to identify versatile solvents despite biochemical variability. Multiple solvents were used for sample extraction prior to analysis by proton NMR and liquid chromatography-mass spectrometry (LC-MS) for multiple botanicals including Camellia sinensis, Cannabis sativa, Myrciaria dubia, Sambucus nigra, Zingiber officinale, Curcuma longa, Silybum marianum, Vaccinium macrocarpon, and Prunus cerasus. Comparisons were normalized by total intensity; deuterated methanol aids NMR lock but is not required for LC-MS. Hierarchical clustering analysis (HCA) evaluated solvent efficacy. Methanol-deuterium oxide (1:1) was the most effective extraction method, yielding 155 NMR spectral metabolite variables for Camellia sinensis, while methanol (90% CH3OH + 10% CD3OD) produced 198 for Cannabis sativa and 167 for Myrciaria dubia, with 11, 9, and 28 assigned metabolites, respectively. LC-MS detected 121 metabolites in Myrciaria dubia in methanol as the most effective extraction method. Methanol (10% deuterated) is the most effective extraction method for comprehensive metabolite fingerprinting using NMR and LC-MS protocols because it provides the broadest metabolite coverage. This study advances fit-for-purpose methods to qualify suppliers of botanical ingredients in food and NHP quality control programs.

Keywords:  botanical authentication; chemotaxonomy; liquid chromatography–mass spectrometry; metabolite fingerprinting; nuclear magnetic resonance; solvent extraction

DOI:  https://doi.org/10.3390/molecules30163379
Foods. 2025 Aug 08. pii: 2768. [Epub ahead of print]14(16):

Development and Validation of QuEChERS Extraction Coupled with Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry for the Detection of Nine Macrolides in Fish Products.

Changhua Sun, Yue Ma, Jia Yang, Xubin Lu, Shuai Wang, Xiangfeng Zheng, Zhenquan Yang, Li Xu, Bo Wang.

  Veterinary drug residues in aquatic products are often overlooked, yet they pose significant environmental risks and potential threats to human health. In this study, a rapid and sensitive analytical method was developed for the simultaneous determination of nine commonly used macrolide antibiotics in largemouth bass (Micropterus salmoides) muscle using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Sample extraction was performed using 80% acetonitrile in water, followed by purification with Cleanert MAS-Q cartridges. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C18 column (50 mm × 2.1 mm; 1.7 μm), equipped with a Waters VanGuardTM BEH C18 guard column (1.7 μm), using a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Mass spectrometric detection was conducted in positive electrospray ionization mode (ESI+) using multiple reaction monitoring (MRM). The method demonstrated excellent linearity in the concentration range of 0.2-30 ng/mL, with determination coefficients (R2) exceeding 0.9980 for all analytes. Average recoveries ranged from 89.3% to 108.4%, with intraday and interday relative standard deviations (RSDs) of 2.9-11.6% and 4.1-12.5%, respectively. The limits of detection (LOD) and quantification (LOQ) for largemouth bass muscle were determined to be 0.4 μg/kg and 2.0 μg/kg, respectively. The decision limits (CCα) and detection capabilities (CCβ) ranged from 2.13 to 215.71 μg/kg and 2.22 to 231.42 μg/kg, respectively. The developed method was successfully applied to the quantitative analysis of macrolide residues in 20 largemouth bass samples collected from local markets.

Keywords:  QuEChERS; UHPLC-MS/MS; aquatic product safety; macrolides; quantitative analysis

DOI:  https://doi.org/10.3390/foods14162768
Anal Chem. 2025 Aug 25.

Increasing the Scale of the Mass Spectrometry Query Language Compendium with Explainable AI.

Thomas V Harwood, Mingxun Wang, Trent R Northen, Benjamin P Bowen.

A significant bottleneck in metabolomics data interpretation is the effective use of domain knowledge to assign structural information based on fragmentation patterns. The mass spectrometry query language (MassQL) aims to make this process accessible and applicable across multiple analysis platforms. While advanced computational methods are capable of predicting compound structures from fragmentation data, AI/ML approaches often rely on complex, opaque criteria that are difficult to interpret or modify. As a result, their predictive patterns cannot be readily translated into human-readable rules, such as those used in MassQL. In this study, we introduce ChemEcho, a machine learning embedding method that converts tandem mass spectrometry data into sparse feature vectors containing peak and neutral mass subformulae to enhance explainable AI/ML-based methods. An advantage of this approach is that decision trees trained using these feature vectors can be directly translated to MassQL. Using a battery of decision trees trained using ChemEcho embeddings to predict molecular attributes, we generated over 1500 MassQL queries for 765 molecular features and evaluated their precision and recall. From these queries, the 50 highest-performing queries were integrated into the MassQL compendium. This set of generated MassQL queries included environmentally and biologically relevant classes such as PFAS and molecules containing phosphate or sulfate substructures. To illustrate the impact these queries would have on a typical metabolomics experiment, these MassQL queries were applied to a public metabolomics data set─resulting in a marked increase in the structural information derived from tandem mass spectra. Access and reuse of these queries is expected to enhance structural annotation in untargeted experiments, leading to more specific claims and advancing many applications in metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.5c02591
J AOAC Int. 2025 Aug 22. pii: qsaf079. [Epub ahead of print]

High-Throughput Quantification of Pesticide Residues in Complex Matrix (Chilli Powder) Using Liquid Chromatography Tandem Mass Spectrometry: Inter- and Intra-Day Validation.

Raviraj Chandrakant Shinde, Macky Suraliwala, Dharmendra Kumar, Sagar Atugade, Pandit Shiragave.

BACKGROUND: Chilli powder is a widely consumed spice, however during cultivation of chilli crops are often subjected to pesticide treatments to control pests and diseases. Thus, monitoring pesticide residues in such matrices is crucial for food safety and to comply with national as well as international regulations for export/import purposes. However, accurate analysis of pesticide residue in chilli powder is challenging due to its complex nature.
OBJECTIVE: To develop and validate a high-throughput LC-MS/MS method for the quantification of multiclass pesticide residues in a complex chilli powder matrix.
METHODS: The acetonitrile-based extraction method was optimized for chilli powder samples. The targeted analytes were separated using reverse-phase liquid chromatography and detected using tandem mass spectrometry. Validation was conducted following SANTE guideline to comply with regulatory requirements.
RESULTS: The method with an optimised sample preparation workflow demonstrated a lower matrix effect of < 35% for the target pesticides. The LOQ was determined to be 0.005 mg/kg for 135 analytes, with recovery ranging from 70 to 110%, and intra-day and inter-day precision (%RSD) were below 15%. Analysis of market/incurred samples and measurement uncertainty further provided more confidence on the method performance.
CONCLUSIONS: The developed LC-MS/MS method provides a robust, sensitive, and high-throughput approach for the quantification of pesticide residues in complex chilli powder. Its intra- and inter-day validation confirms suitability for routine analysis in food safety laboratories.
HIGHLIGHTS: A high-throughput LC-MS/MS method is developed for pesticide analysis in complex chilli powder. The method was validated according to SANTE 11312/2021-v2 with excellent precision and accuracy. Suitable for routine food safety monitoring of wide range of pesticide residues in a testing laboratory.

DOI: https://doi.org/10.1093/jaoacint/qsaf079
Anal Chem. 2025 Aug 26.

QC4Metabolomics: Real-Time and Retrospective Quality Control of Metabolomics Data.

Jan Stanstrup, Lars Ove Dragsted.

The ability to answer complex biological questions in metabolomics relies on the acquisition of high-quality data. However, due to the complex nature of liquid chromatography-mass spectrometry acquisition, data quality checks are often not done comprehensively and only at the postprocessing step. This can be too late to mitigate analytical problems such as loss of m/z calibration, retention time drift and severe ion suppression. It is often not practically or economically feasible to reanalyze samples, and interpretation of the acquired compromised data, if at all possible, is limited, despite the considerable expenses incurred to obtain them. We therefore introduce QC4Metabolomics, a real-time quality control monitoring software for untargeted metabolomics data. QC4Metabolomics monitors files as they are acquired or retrospectively by tracking any user-defined compound(s) and extracting diagnostic information such as observed m/z, retention time, intensity and peak shape, and presents the results on a web dashboard. QC4Metabolomics also monitors the levels of common or user-defined contaminants. We report herein real-world examples where QC4Metabolomics easily reveals analytical problems retrospectively that could have been immediately addressed with real-time monitoring, so that the samples would have been analyzed without any quality control issues. The Shiny app is available as open-source code at https://github.com/stanstrup/QC4Metabolomics. Docker images and a docker-compose setup file are also provided for easy deployment, along with demo data. The documentation can be found at https://stanstrup.github.io/QC4Metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.4c07078
Sci Rep. 2025 Aug 22. 15(1): 30902

Development and validation of a green/blue UHPLC-MS/MS method for trace pharmaceutical monitoring.

Marta Koziarska, Magdalena Strzebońska, Ewa Szalińska.

  In response to the growing concerns regarding pharmaceutical contamination of our aquatic systems, targeted actions are being implemented to align with the recommendations of the European Commission. However, a challenge lies in finding effective, accurate, and green chemistry-compliant methods for analyzing these compounds in complex matrices. This study introduces a highly sensitive and sustainable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneously determining carbamazepine, caffeine, and ibuprofen in water and wastewater. This method exhibits impressive advantages: exceptional sensitivity, high selectivity, and an economical sample preparation strategy resulting from the absence of an evaporation step after solid-phase extraction (SPE), as well as a short analysis time (10 min). Following the International Council for Harmonization (ICH) guidelines Q2(R2), the developed and validated method proved to be specific, linear (correlation coefficients ≥ 0.999), precise (RSD < 5.0%), and accurate (recovery rates ranging from 77 to 160%). The limits of detection were 300 ng/L for caffeine, 200 ng/L for ibuprofen, and 100 ng/L for carbamazepine, respectively. The limits of quantification (LOQs) were 1000 ng/L for caffeine, 600 ng/L for ibuprofen, and 300 ng/L for carbamazepine. The advanced UHPLC-MS/MS method presented in this article constitutes a green and blue analytical technique for the precise detection and quantification of trace levels of pharmaceutical contaminants in aquatic environments. This method has been validated and exemplified using a case study from the Kraków area, highlighting its high efficiency, reliability, and minimal environmental impact. This approach aligns with the concept of sustainable analytics, combining ecological aspects with high-quality results. This study is therefore crucial for the effective monitoring of pollutants, the assessment of environmental and health risks, and ensuring water quality.

Keywords:  Green analytical chemistry; Method validation; Pharmaceuticals; SPE; UHPLC-MS/MS technique; Water

DOI:  https://doi.org/10.1038/s41598-025-15614-4
Molecules. 2025 Aug 21. pii: 3443. [Epub ahead of print]30(16):

Reversed-Phase (RP) and Hydrophilic Interaction (HILIC) Separation Mechanisms for the Assay of Nicotine and E-Cigarette Liquids.

Răzvan Moisi, Mircea-Alexandru Comănescu, Andrei-Valentin Medvedovici.

  Nicotine is a highly used addictive substance that has recently also become available through electronic cigarettes. Here we present a study of nicotine from e-cigarette liquids through reversed-phase (RP) and hydrophilic interaction (HILIC) liquid chromatography. Multiple aqueous mobile-phase additives are considered for the RP mechanism, focusing on chaotropic agents, mobile-phase concentrations and mixing ratios, and column temperature. Sample preparation was conducted by toluene liquid-liquid extraction of e-cigarette liquids diluted with aqueous 25 mM NaHCO3/Na2CO3. Optimal RP results for retention and peak symmetry were obtained using aqueous 0.1% formic acid and 20 mM ammonium hexafluorophosphate with 0.1% formic acid in acetonitrile, using a gradient profile with a C18 column, exploited at 40 °C and a 1.5 mL/min flow rate. A dilute-and-shoot alternative with automated flow reversal after isocratic elution is presented. For HILIC, aqueous 100 mM ammonium formate and 0.1% formic acid in acetonitrile were used as mobile-phase components, using a gradient profile, on a Thermo Scientific™ Acclaim™ Mixed-Mode HILIC-1 column, operated at 25 °C with a 1 mL/min flow rate. UV detection was at 260 nm. Absolute limits of quantitation in the 1 μg/mL range were obtained for all tested alternatives, with 1 μL injection volumes.

Keywords:  HILIC; chaotropic agents; ion pairing; reversed phase

DOI:  https://doi.org/10.3390/molecules30163443
Anal Bioanal Chem. 2025 Aug 21.

Analysis of polychlorinated alkanes in food by liquid chromatography-tandem mass spectrometry.

Ingus Perkons, Laura Lazdina, Dzintars Zacs.

  Polychlorinated alkanes (PCAs), the principal constituents of chlorinated-paraffin technical mixtures, are persistent, bioaccumulative pollutants that raise growing toxicological concern. Due to their complexity, PCA analysis in food remains analytically challenging, predominantly relying on high-resolution mass spectrometry applications. This study aimed to develop and validate a more accessible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying PCA-C10-17 in food commodities. Six reversed-phase columns were evaluated during the study, and phenyl-hexyl and biphenyl stationary phases provided superior separation of critical isobaric PCA homologues. Ammonium acetate (5 mM) was used as a mobile phase additive to promote the formation of acetate adducts, enhancing selectivity in MS/MS settings by minimizing the impact of deprotonated species on product-ion spectra. Validation experiments conducted using fortified samples demonstrated satisfactory recoveries (PCA-C10-13, 88%; PCA-C14-17, 121%; and PCA-C10-17, 103%). Comparative analyses using six interlaboratory test materials and a certified fish matrix reference material confirmed the method's accuracy. All z-scores for PCA-C10-13 were ≤|2|, and only 2 results for PCA-C14-17 were in the questionable range (|z|= 2-3). In the certified reference material, measured values for PCA-C10-13 were within the certified range, while those for PCA-C14-17 were near its lower boundary. The developed method was compared to the conventional high-resolution mass spectrometry, showing a strong agreement between the results of both instrumental setups. These results establish this LC-MS/MS protocol as an accessible and reliable alternative to PCA monitoring within food safety and regulatory frameworks.

Keywords:  Chlorinated paraffins; Food safety; Liquid chromatography; Persistent organic pollutants; Polychlorinated alkanes; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/s00216-025-06070-0
Anal Chem. 2025 Sep 02. 97(34): 18621-18629

Exploring Metal-Organic Framework-Coated Blades for Direct and High-Throughput Screening Analysis of Complex Biological Matrices.

Isaac Negrín-Santamaría, María J Trujillo-Rodríguez, Encarnación Moyano, Juan H Ayala, Olatz Zuloaga, Jorge Pasán, Verónica Pino, Juan F Ayala-Cabrera.

Ambient ionization mass spectrometry (AIMS) techniques have become an emerging approach over the last years due to their simplicity, permitting high-throughput sample analysis by directly coupling with mass spectrometry while ensuring short analysis times. Among AIMS techniques, coated-blade spray (CBS) has stood out, as it ensures a significant enhancement of overall sensitivity, undoubtedly useful for human biofluid analysis. In parallel, the incorporation of advanced smart materials, such as metal-organic frameworks (MOFs), into analytical devices is increasing due to their outstanding ability to efficiently trap target analytes, such as industrial chemicals with diverse functionalities and polarities. This study integrates neat MOFs in CBS devices, without the need for any composite or additional materials, through a simple and mild strategy and shows their use in the determination of xenobiotics present in human urine samples. Moreover, a suspect screening workflow by high-resolution mass spectrometry (HRMS) has been developed for the first time to extend the chemical coverage of AIMS techniques. This simultaneous approach ensures a proper analytical quality performance, achieving limits of quantification (LOQs) down to 0.1 ng·mL-1 despite requiring only 8 min for the entire procedure.

DOI: https://doi.org/10.1021/acs.analchem.5c02782
Ther Drug Monit. 2025 Aug 26.

Simultaneous Determination of Osimertinib and its Metabolites in Human Plasma for Clinical Applications Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Jingjing Wu, Liuxian Guo, Guojin Zhou, Yawen Wang, Ke-Jing Tang, Jing Xu, Jie Chen.

   BACKGROUND: Osimertinib, the first-line treatment for nonsmall cell lung cancer, has 2 main active metabolites, AZ5104 and AZ7550, which demonstrate potency against wild-type epidermal growth factor receptor mutations. In this study, a simple and sensitive analytical method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/mass spectrometry) was developed and validated to simultaneously quantify osimertinib and its active metabolites in human plasma.
METHODS: Plasma samples were subjected to protein precipitation with zinc sulfate solution and acetonitrile using osimertinib-13C, d3 as an internal standard. The separation was performed on a ThermoScientific Hypersil GOLD C18 column (4.6 × 50 mm, 5 μm) with a mobile phase composed of 0.1% formic acid water-acetonitrile through gradient elution. The compounds were monitored using electrospray ionization in the positive mode with multiple reaction monitoring.
RESULTS: Excellent linearity of calibration curves was observed over the ranges of 10-1000 ng·mL-1 for osimertinib, and 1.5-120 ng·mL-1 for AZ7550 and AZ5104 in human plasma. Both within-run and between-run accuracies were within ±15%, with the coefficient of variation <15%.
CONCLUSIONS: The method met all the validation criteria and was successfully applied to clinical samples from patients with nonsmall cell lung cancer. Furthermore, the correlation between steady-state trough concentrations of osimertinib, AZ5104, and AZ7550 and patients' baseline characteristics were explored. Women exhibited a significantly higher Cmin, ss for AZ7550 (P = 0.023). In addition, body mass index was associated with the Cmin, ss of both AZ5104 (P = 0.047) and AZ7550 (P = 0.001).

Keywords:  HPLC–MS/MS; epidermal growth factor receptor–tyrosine kinase inhibitors; nonsmall cell lung cancer; osimertinib; therapeutic drug monitoring

DOI:  https://doi.org/10.1097/FTD.0000000000001355
Nat Commun. 2025 Aug 26. 16(1): 7934

Spatial isotope deep tracing deciphers inter-tissue metabolic crosstalk.

Xinzhu Li, Ying Zhu, Ting Li, Xinyi Tu, Shiyu Zhu, Lingzhi Wang, Fei Li, Chenglong Sun, Xin Li, Haiyi Zhao, Tang Tang, Qingce Zang, Ruiping Zhang, Zeper Abliz.

Organs collaborate to maintain metabolic homeostasis in mammals. Spatial metabolomics makes strides in profiling the metabolic landscape, yet can not directly inspect the metabolic crosstalk between tissues. Here, we introduce an approach to comprehensively trace the metabolic fate of 13C-nutrients within the body and present a robust computational tool, MSITracer, to deep-probe metabolic activity in a spatial manner. By discerning spatial distribution differences between isotopically labeled metabolites from ambient mass spectrometry imaging-based isotope tracing data, this approach empowers us to characterize fatty acid metabolic crosstalk between the liver and heart, as well as glutamine metabolic exchange across the kidney, liver, and brain. Moreover, we disclose that tumor burden significantly influences the host's hexosamine biosynthesis pathway, and that the glucose-derived glutamine released from the lung as a potential source for tumor glutamate synthesis. The developed approach facilitates the systematic characterization of metabolic activity in situ and the interpretation of tissue metabolic communications in living organisms.

DOI: https://doi.org/10.1038/s41467-025-63243-2
J Pharm Biomed Anal. 2025 Aug 18. pii: S0731-7085(25)00465-0. [Epub ahead of print]266 117124

Advances and challenges in sample preparation and analytical methods for water-soluble vitamins: Application in food, clinical, pharmaceutical samples.

Tikeshwari, Kamlesh Shrivas, Khushali Tandey, Anuradha Sharma, Nagendra Kumar Chandrawanshi, Kallol K Ghosh.

  This review provides a comprehensive analysis of advances and challenges in the sample preparation and analytical methods for determining water-soluble vitamins (WSVs) in foods, clinical, and pharmaceutical samples. WSVs, including vitamin C and B-complex vitamins, are essential for various physiological functions such as energy metabolism, immune response, and neurological health. The accurate analysis of these vitamins is crucial for ensuring nutritional adequacy and preventing deficiencies. The paper explores the different separation and extraction techniques, such as acid and enzymatic hydrolysis, solid-phase extraction (SPE), ultrasound-assisted extraction (UAE), and Soxhlet extraction, highlighting their strengths and limitations. Analytical techniques like spectrophotometry, fluorometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and electrochemical methods are reviewed for their applicability in quantifying the WSVs. The review also addresses the challenges in WSV analysis in complex sample matrices, including vitamin stability, matrix effects, and method standardization, which impact the reliability of results. Furthermore, the revolutionary role of nanomaterials, particularly nanoparticles and quantum dots, in enhancing sensitivity for vitamin detection through colorimetric and fluorometric methods is discussed. Finally, different sample preparation and analytical techniques are employed for quantitative measurement of WSVs in foods (vegetables, fruits, and grains) clinical (urine, plasma, tears), pharmaceuticals (syrups, drugs) and metabolic studies. The review concludes by emphasizing the significance of these methodologies in improving the accuracy of WSV analysis, contributing valuable knowledge to the fields of nutrition, clinical and analytical chemistry and supporting public health strategies for preventing vitamin deficiencies.

Keywords:  Analytical methods; Clinicals; Foods; Pharmaceuticals; Sample preparation; Water-soluble vitamins

DOI:  https://doi.org/10.1016/j.jpba.2025.117124
Shokuhin Eiseigaku Zasshi. 2025 ;66(3): 51-55

[Validation Study on a Rapid and Low-priced Method for Determination of Chloroacetic Acids in Mineral Water Products].

Atsuki Ozawa, Takanari Wakayama, Hiroyuki Ohno.

  An LC-MS/MS method for the simultaneous determination of chloroacetic acid, dichloroacetic acid and trichloroacetic acid (chloroacetic acids) in mineral water (MW) products was developed by modifying the Japanese official method to determine chloroacetic acids. To prevent ion suppression or enhancement caused by sample matrix in LC-MS/MS analysis, the Japanese official method comprises the cleanup step of analytes using cation exchange resin column. The step is time-consuming and costly, and thus the modified method adopted small-volume injection instead of it to prevent matrix effects on ionization, which enabled rapid analysis at low cost. A validation study was performed on the method using MW products which vary in the degree of hardness and contents of carbonate. The trueness, repeatability and reproducibility of the method were estimated to be within the ranges of 95.3 to 104.3%, 1.1 to 2.9% and 2.4 to 6.4%, respectively. The values of every performance parameter met the criteria in the guidelines announced by the Ministry of Health, Labour and Welfare of Japan. A recovery study was performed on 30 kinds of MW products to examine the applicability of the method, and the recovery rate of all target substances ranged from 91 to 108%. Therefore, the modified method is considered to be suitable for the determination of chloroacetic acids in MW products.

Keywords:  LC-MS/MS; applicability; chloroacetic acids; mineral water; validation study

DOI:  https://doi.org/10.3358/shokueishi.66.51
J Chromatogr A. 2025 Aug 23. pii: S0021-9673(25)00659-4. [Epub ahead of print]1760 466315

Sensitive analysis of tyrosine kinase inhibitors in plasma using polypropylene fabric-solid phase extraction combined with liquid chromatography-tandem mass spectrometry.

Dong Wang, Mengrong Li, Tingting Dai, Xiangyu Zhang, Di Chen.

  Accurate quantification of tyrosine kinase inhibitors (TKIs) in plasma is essential for therapeutic drug monitoring (TDM). This study presents a novel solid phase extraction (SPE) method employing hydrophobic polypropylene fabric (PPF)-a fibrous material derived from oil-absorbent pads-as the sorbent. The PPF-SPE device was prepared by directly packing fluffed PPF into syringe barrels, eliminating the need for sieve plates and simplifying column assembly. Integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS), the method enabled sensitive determination of eight clinically relevant TKIs (afatinib, alectinib, ceritinib, dasatinib, sunitinib, pazopanib, tamoxifen, and ibrutinib) in plasma. Key parameters, including PPF amount, sample pH, and desorption conditions, were systematically optimized, yielding high extraction recoveries (83.1-96.4 %) and good reproducibility (RSDs<5.5 %). The method showed excellent linearity (R²>0.99), low detection limits (0.017-0.055 ng/mL), and high intra-/inter-day recoveries (85.1 %-102.6 %) and precision (RSDs<5.3 %). Its practical applicability was validated in spiked plasma under a single-blind design, yielding relative errors of -8.2 % to 5.4 %. Compared to traditional SPE methods, PPF-SPE offers significant advantages in operational simplicity, cost-effectiveness, high throughput, and environmental sustainability.

Keywords:  Plasma; Polypropylene fabric; Solid phase extraction; Therapeutic drug monitoring; Tyrosine kinase inhibitors

DOI:  https://doi.org/10.1016/j.chroma.2025.466315
Metabolites. 2025 Aug 05. pii: 531. [Epub ahead of print]15(8):

Exploring 6-aza-2-Thiothymine as a MALDI-MSI Matrix for Spatial Lipidomics of Formalin-Fixed Paraffin-Embedded Clinical Samples.

Natalia Shelly Porto, Simone Serrao, Greta Bindi, Nicole Monza, Claudia Fumagalli, Vanna Denti, Isabella Piga, Andrew Smith.

  Background/Objectives: In recent years, lipids have emerged as critical regulators of different disease processes, being involved in cancer pathogenesis, progression, and outcome. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) has significantly expanded the technology's reach, enabling spatially resolved profiling of lipids directly from tissue, including formalin-fixed paraffin-embedded (FFPE) specimens. In this context, MALDI matrix selection is crucial for lipid extraction and ionization, influencing key aspects such as molecular coverage and sensitivity, especially in such specimens with already depleted lipid content. Thus, in this work, we aim to explore the feasibility of mapping lipid species in FFPE clinical samples with MALDI-MSI using 6-aza-2-thiothymine (ATT) as a matrix of choice. Methods: To do so, ATT performances were first compared to those two other matrices commonly used for lipidomic analyses, 2',5'-dihydroxybenzoic acid (DHB) and Norharmane (NOR), on lipid standards. Results: As a proof-of-concept, we then assessed ATT's performance for the MALDI-MSI analysis of lipids in FFPE brain sections, both in positive and negative ion modes, comparing results with those obtained from other commonly used dual-polarity matrices. In this context, ATT enabled the putative annotation of 98 lipids while maintaining a well-balanced detection of glycerophospholipids (60.2%) and sphingolipids (32.7%) in positive ion mode. It outperformed both DHB and NOR in the identification of glycolipids (3%) and fatty acids (4%). Additionally, ATT exceeded DHB in terms of total lipid count (62 vs. 21) and class diversity and demonstrated performance comparable to NOR in negative ion mode. Moreover, ATT was applied to a FFPE glioblastoma tissue microarray (TMA) evaluating the ability of this matrix to reveal biologically relevant lipid features capable of distinguishing normal brain tissue from glioblastoma regions. Conclusions: Altogether, the results presented in this work suggest that ATT is a suitable matrix for pathology imaging applications, even at higher lateral resolutions of 20 μm, not only for proteomic but also for lipidomic analysis. This could enable the use of the same matrix type for the analysis of both lipids and peptides on the same tissue section, offering a unique strategic advantage for multi-omics studies, while also supporting acquisition in both positive and negative ionization modes.

Keywords:  6-aza-2-thiothymine; FFPE; MALDI-MSI; spatial lipidomics

DOI:  https://doi.org/10.3390/metabo15080531
J Vis Exp. 2025 Aug 08.

Neuropeptide Characterization Workflow from Sampling to Data-Independent Acquisition Mass Spectrometry.

Samuel Okyem, Yanqi Tan, Elena Romanova, Jonathan V Sweedler.

Endogenous neuropeptides are key modulators of brain function, playing critical roles in behavior, stress, pain, and homeostatic regulation, yet their analysis remains difficult. Biologically, they are low in abundance, rapidly degraded, and processed variably from precursor proteins, with expression limited to small, localized cell populations. Technically, their detection is complicated by a wide dynamic range, diverse post-translational modifications, and sparse signals in mass spectrometry datasets. This protocol outlines a comprehensive workflow for neuropeptide analysis in Rattus norvegicus brain tissue using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry (MS) on a timsTOF platform. Following optimized brain sample preparation, including dissection, peptide extraction and clean-up, nano liquid chromatography (LC)-MS is performed with ion mobility gas-phase fractionation to improve detection sensitivity and accuracy. The DDA-generated spectral library supports DIA-based quantification in Skyline, enabling high-confidence MS2-level measurements. This integrated workflow increases neuropeptide coverage and enhances quantitative reproducibility, providing a robust platform for studying neuropeptides in complex brain tissue.

DOI: https://doi.org/10.3791/68741
Talanta. 2025 Aug 22. pii: S0039-9140(25)01219-6. [Epub ahead of print]297(Pt B): 128728

Automated single-cell analysis by single-cell side-orifice flow cytometry chain electrospray ionization mass spectrometry.

Yu Wang, Xia Huang, Zhuolin Jin, Yilin Liu, Lu Chen, Bing Xia, Yan Zhou.

  Single-cell mass spectrometry (SCMS) provides a powerful tool for probing cellular heterogeneity and uncovering biological mechanisms that are often obscured at the population level. However, SCMS faces challenges such as short signal duration and limited tandem mass spectrometry (MS/MS) data for metabolite identification, primarily due to the extremely low sample volume. Previously, we developed the chain electrospray ionization for ultra-low-volume sample analysis. In this paper, a novel single-cell side-orifice flow cytometry combined with the chain electrospray ionization mass spectrometry platform was proposed. This platform enables automated single-cell analysis, successfully identifying 486 lipids from five cell lines (A549, HEK-293T, HCCLM3, MHCC97-H, and SK-HEP-1). Principal component analysis-linear discriminant analysis (PCA-LDA) was employed to accurately differentiate these cell types based on the identified lipid profiles. The platform demonstrates direct, rapid, and high-coverage analysis of single-cell biomolecules, even at extremely low volumes. The novel SCMS not only enhances our understanding of cellular heterogeneity but also holds the potential for identifying disease biomarkers and exploring metabolic pathways in single cells.

Keywords:  Ambient mass spectrometry; Chain electrospray ionization; Side-orifice flow cytometry; Single-cell analysis

DOI:  https://doi.org/10.1016/j.talanta.2025.128728
J Chromatogr A. 2025 Aug 18. pii: S0021-9673(25)00647-8. [Epub ahead of print]1760 466303

HPLC-MS/MS-based detection of Pseudomonas aeruginosa secondary metabolites and their application in metabolomics.

Dan Ran, Ben Li, Hui Wang, Lijian Leng, Jijun Qin, Feifei Zhong, Lu Wang, Lalan Liu, Lili Hu, Haizhi Wu, Shiyu Tian, Hao He, Afang Liu.

  An HPLC-MS/MS method was developed for determining Pseudomonas aeruginosa metabolites in culture media and cosmetics. The samples were dispersed in saturated sodium chloride, purified by C18-SPE after acetonitrile extraction, filtered through a PTFE membrane, and then analyzed. The mobile phase system used was 0.1 % formic acid-methanol. Positive and negative ions are scanned simultaneously, and multiple reaction monitoring (MRM) mode was used for detection. The results revealed that the linear correlation coefficients (r) of the 17 metabolic substances were all greater than 0.99 within the linear range of 1-100 μg/L. Most of the substances had matrix inhibition effect. The recoveries ranged from 85.1 to 114.7 %, and the relative standard deviations (RSD) ranged from 1.3 to 13.3 %. The LOD and LOQ were set as 0.05 mg/kg and 0.1mg/kg respectively. The culture metabolism of Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas fluorescens were analyzed. Pyocyanin, 2-heptyl-4-quinolone (HHQ), phenazine-1-carboxylic acid (PCA) and 1-hydroxyphenazine were found to be unique metabolites of Pseudomonas aeruginosa. The actual sample determination results shows that the metabolic residual risk of Pseudomonas aeruginosa in cosmetics is low. The simulation sample analysis results indicate that cosmetics can inhibit the metabolism of Pseudomonas aeruginosa. The HPLC-MS/MS method demonstrated high sensitivity, strong specificity, excellent selectivity, good accuracy and convenient operation. This study can provide technical support for the applied research on Pseudomonas aeruginosa metabolism and risk screening of residual metabolites in cosmetics. This analytical approach has significant implications for biometabolomics studies, microbial identification, and clinical medicine research.

Keywords:  Biomarker; Cosmetics; Culture media; HPLC-MS/MS; Metabolites; Microbial metabolomics; Residue detection

DOI:  https://doi.org/10.1016/j.chroma.2025.466303
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2025 Aug 20. 1-16

A confirmatory analytical method for post-harvest fungicides in perishable vegetables and fruits using liquid chromatography tandem mass spectrometry.

Keiko Iwakoshi, Hiroyuki Miyakawa, Megumi Hasebe, Ayaka Matsuda, Kenji Otsuka.

  In this study, we developed a simultaneous analysis method for eight post-harvest fungicides commonly used on imported fruits and vegetables using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). The tested fungicides included azoxystrobin, imazalil, propiconazole, pyrimethanil, thiabendazole, difenoconazole, ortho-phenylphenol and fludioxonil. The proposed method employed specialised solid-phase extraction (SPE) columns tailored for these fungicides, streamlining sample preparation and improving our previously developed multi-residue pesticide and preservative analysis techniques. During analysis, samples were extracted with 20 mL of acetonitrile using a ceramic homogeniser. The extract solutions were then purified using two SPE columns before LC-MS/MS analysis. The method demonstrated good linearity, with limits of quantification of 10 µg/kg. During validation, recoveries for the eight fungicides ranged from 73.2% to 105.3%, with relative standard deviation of repeatability values between 0.9% and 17.2% and within-laboratory reproducibility values between 1.9% and 24.7%. Thus, this method meets the Japanese validation guideline criteria, indicating high reproducibility. This method is superior to existing approaches. It reliably analyses a wide range of vegetables and fruits, with a quantification limit of 10 µg/kg. This method can be fully utilised for routine monitoring of fungicides in food safety management.

Keywords:  fungicides; high-performance liquid chromatography with tandem mass spectrometry; perishable vegetables and fruits; solid-phase extraction

DOI:  https://doi.org/10.1080/19440049.2025.2544328
Methods Mol Biol. 2025 ;2958 1-15

Lipidomic Profiling of Isolated Nuclei from Mice and Cultured Cells.

Nityanand Bolshette, Hanmei Bao, Xianlin Han, Gad Asher.

  Lipidomic methodologies, in conjunction with biochemical isolation of intracellular organelles such as the nucleus, have identified a wide variety of nuclear lipid species. The nuclear lipid composition, for example, exhibits daily changes and is controlled both by the circadian clock and feeding behavior. However, the functions of many of these lipids in the nucleus remain elusive. In this chapter we describe in detail the procedure for nuclear lipidomics; from biochemical isolation of nuclei from mouse liver and cultured cells to the unbiased lipid identification and quantification using Multi-Dimensional Mass Spectrometry Shotgun Lipidomics (MDMS-SL). The approach described herein is applicable with some modifications for different tissues and various animal models, as well as a wide variety of cultured cells. It is expected to yield new insights into various aspects of nuclear lipid composition and function in the future.

Keywords:  Cultured cells; Lipid metabolism; Liver; Mass spectrometry; Mouse; Nuclei; Shotgun lipidomics

DOI:  https://doi.org/10.1007/978-1-0716-4714-1_1
Mass Spectrom Rev. 2025 Aug 21.

Analytical Approaches to Address Challenges in the Analysis of Cannabinoids in Vascular Matrices Using Mass Spectrometry.

Radwa Mahmoud, Robert B Laprairie, Anas El-Aneed.

  Phytocannabinoids are bioactive metabolites derived from the Cannabis sativa plant. They have garnered attention due to their recreational uses and therapeutic potential. Although various analytical strategies have been employed for their analysis, mass spectrometry (MS) coupled to chromatographic separation is superior due to its sensitivity and selectivity. Various MS-based strategies, namely Gas chromatography (GC-MS) and liquid chromatography - MS (LC-MS) are reviewed with focus on the analysis of phytocannabinoids in vascular matrices. These include plasma, serum, whole blood, and dried blood spots (DBS). Applications, advantages and challenges associated with each MS strategy in vascular matrices are evaluated and critically discussed. In addition, the review outlines the challenges in DBS spot analysis, such as hematocrit bias, versus plasma/serum and whole blood processing, which involves protein removal, extraction and cleanup steps.

Keywords:  DBS; ESI; GC‐MS; LC‐MS; phytocannabinoids; vascular matrices

DOI:  https://doi.org/10.1002/mas.70005
J Pharm Biomed Anal. 2025 Aug 14. pii: S0731-7085(25)00455-8. [Epub ahead of print]266 117114

An improved pretreatment method for efficient detection of antibiotic residues in aquatic products by LC-MS/MS.

Yu Qin, Shenping Zhang, Jingwen Shi, Heng Gong.

  Antibiotics are a class of compounds that are effective against bacterial infections and are widely used in aquaculture. Herein, a method based on high performance liquid chromatography coupled with tandem mass spectrometry was developed to detect nine classes of 116 antibiotics in aquatic products. The antibiotics were extracted in two steps using 1 % formic acid-acetonitrile with 0.1 mol/L Na2EDTA-Mcllvain buffer solution and acetonitrile, purified using Captiva EMR-Lipid, and separated with an ACQUITY UPLC®HSS T3 column (100 mm × 2.1 mm, 3.5 μm) using acetonitrile and 0.1 % formic acid with 5 mM ammonium acetate as mobile phase for gradient elution. Calibration curve from 0.10 to 100 ng/mL exhibited good correlation coefficient (R2>0.99) for quantification. The analytical performance of the developed method was satisfactory, with limits of quantification ≤ 5.00 ng/g for detecting of antibiotics in aquatic products. The recoveries of the 116 antibiotics ranged from 60.57 % to 127.33 %, with precision (expressed as relative standard deviation) of < 30.20 %. All parameters showed acceptable values and conformed to the Commission Decision 2021/808/EC criteria. When compared with other techniques, the developed method meets the quantitative requirements of high-throughput analysis and can simultaneously analyze 116 antibiotics in aquatic products.

Keywords:  Antibiotics residues; Aquatic products; Captiva EMR-Lipid; LC-MS/MS; Two-step extraction

DOI:  https://doi.org/10.1016/j.jpba.2025.117114
Bioinformatics. 2025 Aug 21. pii: btaf463. [Epub ahead of print]

MultiNMRFit: A software to fit 1D and pseudo-2D NMR spectra.

Pierre Millard, Loïc Le Grégam, Svetlana Dubiley, Valeria Gabrielli, Thomas Gosselin-Monplaisir, Guy Lippens, Cyril Charlier.

MOTIVATION: Nuclear Magnetic Resonance (NMR) is widely used for quantitative analysis of metabolic systems. Accurate extraction of NMR signal parameters-such as chemical shift, intensity, coupling constants, and linewidth-is essential for obtaining information on the structure, concentration, and isotopic composition of metabolites.
RESULTS: We present MultiNMRFit, an open-source software designed for high-throughput analysis of one-dimensional NMR spectra, whether acquired individually or as pseudo-2D experiments. MultiNMRFit extracts signal parameters (e.g. intensity, area, chemical shift, and coupling constants) by fitting the experimental spectra using built-in or user-defined signal models that account for multiplicity, providing high flexibility along with robust and reproducible results. The software is accessible both as a Python library and via a graphical user interface, enabling intuitive use by end-users without computational expertise. We demonstrate the robustness and flexibility of MultiNMRFit on 1H, 13C and 31P NMR datasets collected in metabolomics and isotope labeling studies.
AVAILABILITY: MultiNMRFit is implemented in Python 3 and was tested on Unix, Windows, and MacOS platforms. The source code and the documentation are freely distributed under GPL3 license at https://github.com/NMRTeamTBI/MultiNMRFit/ and https://multinmrfit.readthedocs.io, respectively.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btaf463
Talanta. 2025 Aug 23. pii: S0039-9140(25)01227-5. [Epub ahead of print]297(Pt B): 128736

Stable isotope-labeled paper spray mass spectrometry enables high-sensitivity profiling of protease activity.

Zhiqing Zhou, Weiqing Wang, Xiunan Zhai, Miaomiao Qin, Zhao Wang, Yanmei Yang.

  Proteases play critical roles in disease pathogenesis, and their activity detection is essential in the diagnostic processes for inflammatory disorders, cancers, and neurodegenerative diseases. Conventional approaches for protease detection, such as traditional mass spectrometry detection methods, are susceptible to interference from complex matrices. In recent decades, stable isotope-labeled internal standards (IS) have been widely employed as an effective strategy to mitigate matrix effects. However, existing stable isotope IS face challenges, including difficulty in finding suitable compounds, complex synthesis processes, and high costs. In this study, we demonstrated a novel class of stable isotope labeling reagents and applied them to construct stable isotope-labeled nanoprobes (SIL-nanoprobes). These nanoprobes allow direct, sensitive and specific quantitation of calpain-2 (CAPN2) protease activity in serum using paper spray mass spectrometry (PS-MS). The CAPN2-specific recognition peptide was covalently conjugated to AuNPs, followed by labeling with stable isotope reagents via click chemistry. Upon CAPN2 cleavage, these SIL-nanoprobes generated characteristic MS signal pairs from light and heavy isotope-labeled fragments, enabling precise MS detection. Matrix effects were significantly reduced and the detection limit was achieved to 0.3807 ng/mL. By using an internal isotope-labeled standardization approach, our present strategy enables direct and accurate quantification of CAPN2 activity using PS-MS platform, without external procurement. This strategy holds great promise for other types of protease detection systems by substituting the protease-specific peptide substrates.

Keywords:  PS-MS; Protease activity; Stable isotope labeling reagent

DOI:  https://doi.org/10.1016/j.talanta.2025.128736
Anal Chem. 2025 Sep 02. 97(34): 18562-18572

Improvement of Electrospray Ionization Response Linearity and Quantification in Dissolved Organic Matter Using Synthetic Deuterated Internal Standards.

Alexander J Craig, Mustapha A Ganiyu, Lindon W K Moodie, Sofja Tshepelevitsh, Koit Herodes, Heike Simon, Thorsten Dittmar, Jeffrey A Hawkes.

Aquatic dissolved organic matter (DOM) is an ultracomplex mixture of compounds covering a wide range of masses and with an unknown extent of isomeric complexity, making its structural elucidation and quantification highly challenging. Electrospray ionization high-resolution mass spectrometry (ESI-HRMS) has advanced DOM analysis, but accurate concentration determination remains limited by the lack of a response factor correction. Here, we address this limitation by introducing novel deuterated compounds as internal standards that mimic DOM structures. Using a d5-labeled compound free of isobaric interferences in DOM, we assessed ionization suppression in various aquatic sample extracts and improved concentration-based linearity in a coastal DOM reference material. Our results show that deuterated carboxylic acid-rich standards enable "pseudoquantification" by correcting for ionization suppression and instrument drift. Applying this approach, we estimate that DOM consists of 20-30% acids in river, coastal, and deep-ocean reference samples using an Orbitrap system. The same samples were estimated to contain 11-24% acids using 15T FT-ICR-MS, highlighting platform differences. Additionally, we establish a ∼1 ng L-1 feature detection limit for DOM compounds in seawater via a standard LC-MS gradient method. These findings demonstrate that deuterated standards provide a simple, practical way to improve DOM pseudoquantification, enhancing our understanding of its chemical composition in environmental studies.

DOI: https://doi.org/10.1021/acs.analchem.5c02463
Chem Res Toxicol. 2025 Aug 20.

Identification of Anatoxin-a and Related Metabolites in Exposed Mice Samples with a High-Resolution Mass Spectrometry Discovery Workflow.

Taylor J Glattke, Mike A Mojica, Kirsten A Cottrill, Sarah R Lagon, Brenda Ruto, Donna Hill, Brady R Cunningham.

Harmful cyanobacterial blooms (HCBs) are a public health concern and require ongoing surveillance to monitor the negative water quality effects and cyanotoxins associated with these blooms. (+)-Anatoxin-a (ATX) is a potent neurotoxin produced by select cyanobacteria during HCB formation. Many HCB toxins are commonly associated with discolored water; however, ATX can be present in clear water, which results in a high risk of exposure by accidental ingestion for humans and animals. In this work, we used a qualitative, semitargeted liquid chromatography high resolution mass spectrometry (LC-HRMS) method and a discovery data analysis workflow to detect and identify ATX and its predicted mammalian metabolites in urine samples from ATX-dosed mice. Potential compounds were evaluated for identification with product-ion spectral matching to a local library, accurate mass list matching, further data processing and interpretation, and comparison to undosed mice urine samples. As a result, ATX and dihydroanatoxin-a (dhATX) were successfully identified in the dosed mice samples through retention time (RT) and product-ion spectral matching to their respective commercial standards. The positive identification of dhATX suggests its formation as an abundant metabolic product of ATX within mammalian systems. Additionally, multiple chromatographic peaks were observed that matched the exact mass of 3-OH ATX and were further identified by the presence of diagnostic product ions and comparison to a standard synthesized in-house. In total, seven potential ATX metabolites, including dhATX and 3-OH ATX, were detected and characterized in the dosed mice samples. All identified metabolites were either oxidized or reduced forms of ATX, which suggests that oxidation and reduction are the main pathways for endogenous ATX metabolism in mice. These results are among the first reports of metabolic products of ATX in biological samples and provide a metabolic profile of ATX for higher confidence screening for ATX after a suspected exposure event.

DOI: https://doi.org/10.1021/acs.chemrestox.5c00236
Rapid Commun Mass Spectrom. 2025 Dec 15. 39(23): e10127

Stable Nitrogen Isotope Analysis of Amino Acids by Orbitrap Mass Spectrometry: Application for Extraterrestrial Samples.

O M McIntosh, A A Baczynski, M Matney, H L McLain, K K Farnsworth, J P Dworkin, D P Glavin, J E Elsila, H Xie, K H Freeman.

   BACKGROUND: Obtaining isotopic data on soluble organic compounds, such as amino acids, in extraterrestrial samples is crucial for understanding their origins, prebiotic chemistry, and potential contamination. Conventional GC-IRMS requires grams of material to measure isotopic compositions, limiting the analysis of low-concentration organics in meteorites and other astromaterials. We present an Orbitrap-based method optimized for nitrogen isotopic analysis of amino acids.
RESULTS: This method determines δ15N values for picomole quantities (< 150 pmol) with 3‰-8‰ precision and accuracy within 2‰ compared with elemental analysis. Our approach was validated using amino acid enantiomer standards and a CM2 Murchison meteorite sample. The Murchison results demonstrate that comparable precision can be achieved on analytes extracted from a total sample size representing less than 7% of the mass previously required for CSIA analysis of the same meteorite.
SIGNIFICANCE: These results highlight the potential of Orbitrap mass spectrometry for δ15N measurements with less material and lower analyte concentrations. This technique improves our ability to trace the origins and synthetic pathways of amino acids, providing valuable insights into prebiotic chemistry and possible abiotic mechanisms for organic compound formation in primitive solar system materials. Nitrogen isotopes serve as a powerful tool for distinguishing biological from non-biological sources, aiding in the identification of contamination in meteoritic samples and improving the reliability of analyses involving rare extraterrestrial materials.

Keywords:  GC‐Orbitrap‐IRMS; Murchison meteorite; amino acids; carbonaceous chondrites; nitrogen stable isotopes

DOI:  https://doi.org/10.1002/rcm.10127