bims-metlip 2025-06-29 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–06–29
thirty-two papers selected by
Sofia Costa, Matterworks

Validated LC-MS/MS Method for Quantifying the Antiparasitic Nitroimidazole DNDI-0690 in Preclinical Target Site PK/PD Studies.
Development and Validation for the Quantification of Genotoxic Impurity in Mirabegron Drug Substance by LCMS.
Quantitative analysis of ethylene glycol in human serum by liquid chromatography-tandem mass spectrometry with p-toluenesulfonyl isocyanate derivatization.
Assessing the Impact of Measurement Precision on Metabolite Identification Probability in Multidimensional Mass Spectrometry-Based, Reference-Free Metabolomics.
Quantitative analysis of stimulants in whole blood using an evaporation free precipitation salt assisted liquid-liquid extraction (SALLE) sample preparation approach.
Development and validation of an LC-MS/MS method for quantifying a new G protein selective μ-opioid receptor agonist YZJ-4729 and its major metabolite M10 in human plasma to support a phase I study in healthy subjects.
Rapid Enantiomeric Ratio Determination of Multiple Amino Acids Using Ion Mobility-Mass Spectrometry.
Rapid Multi-Omics for Bacterial Identification Using Flow Injection-Ion Mobility-Mass Spectrometry.
Development and Validation of a New LC-MS/MS Method for the Assay of Plasmatic Peripheral Short- and Medium-Chain Fatty Acids for Metabolomics Applications.
Towards Automated Testing of Kynurenine for Point-of-Care Metabolomics.
Quantitation of plasma and urine 3-methoxytyramine using ID‑LC-MS/MS: A candidate reference measurement procedure and its application to evaluate the results from more than 100 routine LC-MS/MS labs in China.
Integrating NMR and MS for Improved Metabolomic Analysis: From Methodologies to Applications.
JESTR: Joint Embedding Space Technique for Ranking Candidate Molecules for the Annotation of Untargeted Metabolomics Data.
Steroidomics via Gas Chromatography-Mass Spectrometry (GC-MS): A Comprehensive Analytical Approach for the Detection of Inborn Errors of Metabolism.
Quantitative approaches for spatial metabolomics with isomer differentiation using surface sampling capillary electrophoresis mass spectrometry.
A novel UHPLC-MS/MS approach for simultaneous quantification of pyrimidine metabolites in human biofluids.
[Determination of 27 perfluoroalkyl substances in pork by ultra-high performance liquid chromatography-tandem mass spectrometry].
Simultaneous Determination of Buspirone, Alprazolam, Clonazepam, Diazepam, and Lorazepam by LC-MS/MS: Evaluation of Greenness Using AGREE Tool.
Isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedures for the quantification of total and free phenytoin in human serum and plasma.
SMQVP: A Web Application for Spatial Metabolomics Quality Visualization and Processing.
An Integrated Platform for High-Throughput Extraction and Mass Spectrometry-Based Quantification of Cholesterol and Sphingosine.
Optimization and validation of the Kairos amino acid Kit for plasma amino acid monitoring in inherited metabolic disorder patients.
Development of an LC-MS/MS method for quantification of colistin and colistin methanesulfonate in human plasma and its application to stability studies and therapeutic drug monitoring.
Simultaneous Biomonitoring of Common Food Carcinogen Acrylamide and its Major Metabolites using Fast Urinary Metabolites Extraction Technique Coupled with LC-MS/MS Analysis.
A Compact Method Specialized for Full Analysis of the Glycan Composition of Jellyfish Mucin Using Limited Degradation Followed by High-Resolution Mass Spectrometry.
Establishment and Validation of Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Aldosterone Quantification in Feline Serum with Reference Interval Determination.
Laser capture microdissection-assisted gas chromatography-triple-quadruple mass spectrometry for spatial metabolic profiling of esophageal squamous cell carcinoma.
Introducing Korea metabolomics data repository (KMAP): bridging Korean metabolomics data to global data sharing infrastructure.
Breath and Blood Metabolomics: A Comparative Study Using SESI-HRMS/MS and UHPLC-ESI-HRMS/MS.
An LC-MS Method to Quantify Rhein and Its Metabolites in Plasma: Application to a Pharmacokinetic Study in Rats.
Quantitative analysis of seven free short-chain fatty acids in infant formula using GC-MS/MS.
High-throughput quantification of butachlor by DART-MS/MS coupled with micro-solid-phase extraction and dual fragmentation to enhance selectivity and sensitivity.

Biomed Chromatogr. 2025 Aug;39(8): e70158

Validated LC-MS/MS Method for Quantifying the Antiparasitic Nitroimidazole DNDI-0690 in Preclinical Target Site PK/PD Studies.

Wietse M Schouten, Katrien van Bocxlaer, Hilde Rosing, Alwin D R Huitema, Jos H Beijnen, Jadel M Kratz, Charles E Mowbray, Thomas P C Dorlo.

  Understanding the target site pharmacokinetics (PK) of the nitroimidazole analog DNDI-0690, a potential drug for the neglected parasitic disease leishmaniasis, is important due to the diversity of infected tissue sites and potential drug penetration variability. An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for quantifying DNDI-0690 in murine biomatrices (plasma, liver, spleen, skin, and skin microdialysate). The method used three protein precipitation sample preparation procedures, tailored for different biomatrices, utilizing a surrogate biomatrix approach. Murine tissues were enzymatically homogenized with a Collagenase A mixture. Chromatographic detection was performed on a C18 column using gradient elution, coupled to a QTRAP6500 quadrupole MS, operating in positive ionization mode. The method demonstrated accurate and precise quantification of all murine biomatrices on the surrogate biomatrix calibration standards, with a high and reproducible total recovery ranging from 75.9% to 94.2% (CV% ≤ 2.5%). Matrix interferences were mitigated with a deuterated internal standard. Stability experiments demonstrated that DNDI-0690 remained stable in all biomatrices under various conditions. This validated UHPLC-MS/MS method was successfully used to quantify DNDI-0690 in a target site murine infection model, demonstrating its suitability for future target site PK studies involving DNDI-0690.

Keywords:  DNDI‐0690; UHPLC‐MS/MS; leishmaniasis; nitroimidazole; target site pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70158
Biomed Chromatogr. 2025 Aug;39(8): e70150

Development and Validation for the Quantification of Genotoxic Impurity in Mirabegron Drug Substance by LCMS.

Haresh B Patel, Rohit H Dave, Sandip Vadariya, Hitin Hirpara.

  The mirabegron drug substance was shown to have potential genotoxic impurity (PGI); hence, it needs to develop the method for the determination of the 2-(2-Nitrophenyl) ethylamine hydrochloride (4-NPE RC-2) PGI at a 30-ppm limit considering the maximum daily dose of 50 mg/day and a 1.5 mg per day TTC limit as per ICH M7 guideline. A simple high-performance liquid chromatography (HPLC) method can be used due to the chromophore nature of the impurity, but due to sample matrix interference on the HPLC method, a selective and specific method was developed using liquid chromatography-mass spectrometry (LC-MS/MS) method. Using an Inertsil ODS 3-V column, gradient elution mode, 0.8 mL per min flow rate, 0.2% formic acid aqueous solution as mobile phase A, and acetonitrile solvent as mobile phase B was used for better chromatographic separation. Triple quadrupole mass detection with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode was used for determination and quantification. For this PGI, the procedure was thoroughly verified with good linearity across the mirabegron test concentration range of 9-45 ppm. Linearity was observed with an exceeding 0.990 correlation coefficient and found accuracy in terms of recovery between 100.0% and 103.0% with below 5.0% relative standard deviation.

Keywords:  LCMS analysis; genotoxic impurities; method development; method validation; mirabegron

DOI:  https://doi.org/10.1002/bmc.70150
Forensic Toxicol. 2025 Jun 24.

Quantitative analysis of ethylene glycol in human serum by liquid chromatography-tandem mass spectrometry with p-toluenesulfonyl isocyanate derivatization.

Shin Ogawa, Ryosuke Shiraki, Kengo Wakigawa, Hidehiko Okazaki, Akira Tsujita, Akinaga Gohda, Toshiro Matsui.

   PURPOSE: Ethylene glycol (EG) is a typical antifreeze compound and a significant analyte in forensic toxicology. Current EG analytical method for biological samples in forensic toxicology employ liquid chromatography-tandem mass spectrometry (LC-MS/MS), however, they exhibit low sensitivity and reliability. Therefore, in this study, we aimed to establish a highly sensitive, selective, and reliable EG assay system for human serum analysis using a hydroxyl derivatization-aided LC-MS/MS technique.
METHODS: p-Toluenesulfonyl isocyanate (PTSI) was applied for precolumn derivatization of EG in human serum, to enhance the sensitivity of LC-MS/MS for EG detection.
RESULTS: The optimal derivatization conditions were 200 µL/mL PTSI in acetonitrile at 25 °C for 10 min. A highly sensitive and reliable LC-MS/MS detection of EG in human serum was achieved, with the calibration curve exhibiting a good linearity (r > 0.999, 10-1000 µg/mL of EG). The proposed PTSI-derivatization-LC-MS/MS method exhibited high reliability (1.4-1.8%) for the intra-day and inter-day repeatability (%RSD), and accuracy (96.7-102.4%), with the limits of detection and quantification in human serum being 0.023 µg/mL (S/N = 3) and 0.077 µg/mL (S/N = 10), respectively.
CONCLUSIONS: A novel PTSI derivatization-aided LC-MS/MS method was developed, offering a highly sensitive, selective, and reliable analytical tool for EG quantification in human serum for forensic toxicology applications.

Keywords:   p-Toluenesulfonyl isocyanate; Derivatization; Ethylene glycol; Human serum; LC–MS/MS

DOI:  https://doi.org/10.1007/s11419-025-00729-0
Anal Chem. 2025 Jun 25.

Assessing the Impact of Measurement Precision on Metabolite Identification Probability in Multidimensional Mass Spectrometry-Based, Reference-Free Metabolomics.

Christine H Chang, Sydney C Schwartz, Alexandria K Im, Kent J Bloodsworth, Bobbie-Jo M Webb-Robertson, Robert G Ewing, Thomas O Metz, Dylan H Ross.

Identification of compounds with minimal ambiguity remains a central challenge in mass spectrometry-based metabolomics. Conventional compound identification relies on comparing analytical signatures (e.g., mass-to-charge ratio, collision cross section, tandem mass spectra) against reference data obtained from measurements of authentic chemical standards. The breadth of annotatable compounds using this approach is necessarily limited by availability of authentic standards, analytical throughput, and resolving power of the separations that underly the measurements. The maturation of computational methods, both theory-driven and artificial intelligence/machine learning-based, for prediction of various molecular properties relevant to multidimensional mass spectrometry measurements has opened the door to a new "reference-free" paradigm of compound annotation. Through augmenting existing reference data for molecular properties with computational predictions, the universe of identifiable chemical species can be expanded significantly beyond its current limits. An unexplored aspect of this novel approach is understanding how to gauge confidence in resulting annotations, especially as the compound search space is expanded. Intuitively, the confidence of a compound annotation is related to the inherent discriminatory power of the molecular properties used for identification, as well as the precision with which the properties are measured or predicted. In this work, we characterize this relationship between measurement precision and identification probability in a systematic and quantitative fashion for a defined region of chemical space that includes organic small molecule metabolites. Importantly, this work establishes a framework for conducting metabolite identification probability analysis that enables others to quantify this relationship for their own compounds and properties of interest.

DOI: https://doi.org/10.1021/acs.analchem.5c01067
Forensic Toxicol. 2025 Jun 24.

Quantitative analysis of stimulants in whole blood using an evaporation free precipitation salt assisted liquid-liquid extraction (SALLE) sample preparation approach.

Jon Stephenson, Joseph Austin, Bailey Carney, Melanie Flater, Skye Mullarkey, Michael Morrison.

   PURPOSE: The increasing prevalence of methamphetamine and cocaine in postmortem toxicology casework has placed significant demands on forensic laboratories. This study introduces and validates a streamlined method using salt assisted liquid-liquid extraction (SALLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to improve the efficiency and reliability of detecting amphetamine-type stimulants (ATS) and cocaine metabolites in forensic toxicology.
METHODS: A new SALLE method was developed to analyze a panel of drugs, including amphetamine, methamphetamine, phentermine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), pseudoephedrine, cocaine, cocaethylene, and benzoylecgonine (BZE). Calibration models, bias, precision, recovery, matrix effects, interferences, limits of detection (LOD) and quantitation (LOQ), dilution integrity, carryover, and sample stability were evaluated following AAFS standard 036 guidelines. The method was applied to over 150 postmortem and human performance toxicology cases and compared with the traditional gas chromatography-mass spectrometry (GC/MS) approach.
RESULTS: The SALLE-LC-MS/MS method exhibited high accuracy, with all analytes meeting bias and precision criteria (< 20%). Percent recovery values exceeded 80%, while matrix effect values (ion suppression/enhancement) remained below 20%. LODs ranged from 5-25 µg/L, and LOQs ranged from 10-50 µg/L across analytes. Processed samples were stable for up to 8 days. Analysis of 150 cases showed strong agreement with the GC/MS method, with average percent differences ranging from 5.4 to 19.4% for most analytes. The new method reduced sample preparation time by 67% and data-processing time by 80%, resulting in overall time savings of 8 h per batch.
CONCLUSIONS: The resulting validated SALLE procedure represents a significant advancement in the analysis of stimulant drugs within forensic toxicology. Its adoption at the Georgia Bureau of Investigation not only addresses current analytical challenges but also sets a precedent for the development of more efficient and reliable methods in the field.

Keywords:  Cocaine; LC–MS/MS (liquid chromatography-tandem mass spectrometry); Methamphetamine; SALLE (salt-assisted liquid–liquid extraction); Stimulants

DOI:  https://doi.org/10.1007/s11419-025-00724-5
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jun 24. pii: S1570-0232(25)00268-5. [Epub ahead of print]1263 124714

Development and validation of an LC-MS/MS method for quantifying a new G protein selective μ-opioid receptor agonist YZJ-4729 and its major metabolite M10 in human plasma to support a phase I study in healthy subjects.

Yufeng Ni, Chenxin Ding, Minlu Cheng, Li Ding.

  The use of conventional opioids that act on μ-receptors is a recognized care agent for moderate-to-severe acute treatment. However, recent attention has shifted to a new class of μ-receptor agonists which can selectively activate the G-protein pathway and thus have better efficacy and fewer side effects. Recently, YZJ-4729 was developed as a new G protein selective μ-opioid receptor agonist. And for its clinical trial investigation usage, a rapid LC-MS/MS method was established for the concurrent measurement of YZJ-4729 and its major metabolite M10 in human plasma. A step involving the precipitation of proteins was used for plasma sample preparation. The chromatography separation was done on a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm, 2.7-μm, Agilent). Gradient elution was performed with 5.0 mM ammonium acetate (NH4Ac) and 0.1 % formic acid (FA) water solution as the mobile phase A and pure acetonitrile (ACN) as the mobile phase B. Detection occurred in the mode of positive ion electrospray ionization through multiple reaction monitoring using deuterium YZJ-4729 (d6-YZJ-4729) as the internal standard. The ionic transitions used were YZJ-4729: m/z 409.3 → 244.2; d6-YZJ-4729: m/z 415.3 → 244.2; M10: m/z 425.3 → 260.2;). The method showed excellent linearity across the ranges of 0.500 to 500 ng/mL for YZJ-4729 and 0.0500 to 50.0 ng/mL for M10. The method was utilized to assess the plasma concentrations of YZJ-4729 and M10 in healthy volunteers after a 30-min intravenous infusion in the phase I clinical study, and the clinical pharmacokinetic profiles of YZJ-4729 and M10 were described.

Keywords:  Clinical pharmacokinetics.; LC-MS/MS.; Metabolite.; Selective μ-opioid receptor agonist.; YZJ-4729

DOI:  https://doi.org/10.1016/j.jchromb.2025.124714
Molecules. 2025 Jun 06. pii: 2497. [Epub ahead of print]30(12):

Rapid Enantiomeric Ratio Determination of Multiple Amino Acids Using Ion Mobility-Mass Spectrometry.

Wenqing Xu, Estelle Rathahao-Paris, Sandra Alves.

  Chiral analysis is becoming increasingly important across various scientific fields, including chemistry, pharmaceuticals, biosciences, and more recently, metabolomics. In this context, a high-resolution and high-throughput method was developed for the simultaneous determination of the enantiomeric ratio (er) of seven pairs of amino acid (AA) enantiomers (Arg, Gln, His, Met, Pro, Tyr, and Trp) using flow injection analysis coupled with ion mobility-mass spectrometry (FIA-IM-MS) technology. Specifically, the Single Ion Mobility Monitoring (SIM2) mode on a TIMS-TofTM instrument enabled the rapid relative quantification of chiral compound mixtures. A linear model accurately described the relationship between enantiomeric ratio and IM-MS response for Arg, Gln, and Pro enantiomers, as evidenced by high R2 values and unbiased residuals. In contrast, non-linear trends were observed for His, Tyr, and Trp, where a quadratic model significantly improved the fit. However, the linear model was retained for Met, despite an R2 of about 0.98, due to its comparable performance and simplicity. Measurement accuracy was confirmed with very good recovery rates for er values of 0.95 and 0.99 across all AAs. Finally, the potential of the FIA-SIM2-MS approach in chiral analysis was demonstrated, particularly its ability to provide a reliable and efficient high-throughput tool for accurate er determination.

Keywords:  chiral analysis; enantiomeric ratio; flow injection analysis; ion mobility-mass spectrometry

DOI:  https://doi.org/10.3390/molecules30122497
Anal Chem. 2025 Jun 24.

Rapid Multi-Omics for Bacterial Identification Using Flow Injection-Ion Mobility-Mass Spectrometry.

Hannah M Hynds, Jana M Carpenter, Kelly M Hines.

The implementation of mass spectrometry (MS) in clinical microbiology has made a significant improvement in the turnaround time from positive culture to identification, but current protein-based approaches can struggle with species-level identification because of the high degree of homology within a genus. However, other MS-based strategies for bacterial identification that are based on lipids and small molecules have shown promise toward species-level identification and detection of specific phenotypes, including those related to antibiotic resistance. We are leveraging rapid gas-phase ion mobility (IM) separations coupled to MS to simultaneously detect the lipids and metabolites in bacterial pathogens. Using flow-injection (FI) rather than liquid chromatography (LC), we instead rely more directly on the structural separation of the IM dimension to resolve features from different biochemical classes and aid in identification. A head-to-head comparison demonstrates that the FI-IM-MS multiomic strategy performs similarly to LC-IM-MS in its ability to distinguish 24 strains of the high-concern ESKAPE pathogens, while shortening overall analysis time from 17 to 2 min per injection. We demonstrate that the IM dimension has excellent stability and reproducibility, which enables extracted IM peak areas to be used in lieu of chromatographic peak areas. Furthermore, the same features that are important for the discrimination of bacterial species and strains are found within both the FI-IM-MS and HILIC-IM-MS data sets. These results showcase the capabilities of mobility-enabled rapid multiomics and open the possibility to detect subtle strain-level differences and resistance phenotypes in bacterial pathogens by including additional classes of biomolecules.

DOI: https://doi.org/10.1021/acs.analchem.5c00417
Metabolites. 2025 Jun 16. pii: 403. [Epub ahead of print]15(6):

Development and Validation of a New LC-MS/MS Method for the Assay of Plasmatic Peripheral Short- and Medium-Chain Fatty Acids for Metabolomics Applications.

Lenard Farczadi, Laura Barcutean, Smaranda Maier, Rodica Balasa, Silvia Imre.

  Background: Short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) are human metabolites which are involved in various biochemical processes and can offer valuable insights and information on various pathological and metabolic issues of patients. Accurate, precise, high-performance bioanalytical methods are important tools in both research and diagnostics of many pathologies, with LC-MS being the most frequently used methodology in modern metabolomics studies. Methods: The current paper describes a complete LC-MS/MS methodology for the accurate quantification of total plasmatic SCFA concentrations in humans using high-resolution QTOF mass spectrometric detection, including sample cleanup, preparation, and derivatization. Results and Conclusions: The method was validated with regard to all relevant parameters (selectivity, sensitivity, accuracy, precision, linearity, recovery, carryover, and reproducibility of sample preparation) according to the current applicable guidelines and tested in an in vivo study to quantify peripheral SCFAs in human patients as biomarkers for gut-brain axis disruption.

Keywords:  LC-MS/MS; multiple sclerosis; plasma; short-chain fatty acids

DOI:  https://doi.org/10.3390/metabo15060403
Methods Protoc. 2025 Jun 01. pii: 56. [Epub ahead of print]8(3):

Towards Automated Testing of Kynurenine for Point-of-Care Metabolomics.

Dipanjan Bhattacharyya, Marcia A LeVatte, David S Wishart.

  Our objective was to develop a simple, low-cost colorimetric assay to detect kynurenine (L-Kyn) in human biofluids, that would be compatible with a point-of-care (POC) system being developed in our lab. Elevated L-Kyn is associated with many pathological conditions. However, current detection methods are expensive, time-consuming, and unsuitable for resource-limited settings. Existing colorimetric L-Kyn assays lack specificity, require unusual reagents, or lack sensitivity, hindering their practical application. Here we report a two-step diazotization-based colorimetric assay that produces a red chromophore upon reaction with L-Kyn. To reduce background interference, we used dilution and anion exchange chromatography for urine samples and acid precipitation for serum samples. The assay detected 5-300 μM L-Kyn in urine (lower limit of detection (LLOD) 1.34 μM) and 5-125 μM L-Kyn in serum (LLOD 1.24 μM). Correlation studies achieved strong linearity (R2 = 0.98 for spiked urine, 0.99 for spiked serum) and were highly correlated (>0.95) to liquid chromatography tandem mass spectrometry (LC-MS/MS) concentrations. Bland-Altman analysis confirmed agreement between L-Kyn assay and LC-MS/MS methods. To our knowledge, this is the first application of a diazotization reaction for L-Kyn quantification at physiologically relevant levels. The assay is now being ported to a low-cost, automated POC biosensor platform.

Keywords:  LC-MS/MS; chemical assay; diazotization; kynurenine; serum; urine

DOI:  https://doi.org/10.3390/mps8030056
Clin Chim Acta. 2025 Jun 21. pii: S0009-8981(25)00310-9. [Epub ahead of print]577 120431

Quantitation of plasma and urine 3-methoxytyramine using ID‑LC-MS/MS: A candidate reference measurement procedure and its application to evaluate the results from more than 100 routine LC-MS/MS labs in China.

Zijia Ma, Xuanchang Bai, Zhenni Liu, Yuhang Deng, Kaiduo Xu, Weiyan Zhou, Chuanbao Zhang.

   BACKGROUND: Accurate measurement of 3-methoxytyramine (3MT) is essential for diagnosing metastatic pheochromocytomas, paragangliomas, and neurodegenerative diseases like Parkinson's disease. Despite the growing use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 3MT quantification, poor comparability persists among laboratories. This study developed an isotope dilution LC-MS/MS-based candidate reference measurement procedure (cRMP) for plasma and urine 3MT measurement and evaluated LC-MS/MS results from over 100 laboratories in China.
METHODS: Plasma/urine samples were spiked with internal standard, followed by protein precipitation and solid-phase extraction. The final extract was analyzed using LC-MS/MS. The cRMP assigned values to four samples (202411-202414), which were sent on dry ice to over 100 laboratories in China for analysis using routine LC-MS/MS methods. Laboratory results were compared to cRMP-assigned target values.
RESULTS: The method validation of cRMP showed good precision, with intra-run and total imprecision ranging from 0.97% to 3.85% and 0.99% to 4.03%, respectively. The relative recovery rate ranged from 99.51% to 104.61%. Significant biases were observed when comparing the measurement results of routine laboratories with the target values assigned by cRMP, and the results from different laboratories were not comparable.
CONCLUSION: Our LC-MS/MS cRMP demonstrates high accuracy, precision, specificity, and sensitivity for 3MT measurements. By utilizing SI-traceable certified reference materials(3MT Cerilliant standard), it ensures robust traceability. This cRMP serves as a high-order standard for establishing traceability, calibrating routine methods, and developing reference materials, supporting the standardization of 3MT assays.

Keywords:  3-methoxytyramine; Consistency; Liquid chromatography-tandem mass spectrometry; Reference measurement procedure; Standardization

DOI:  https://doi.org/10.1016/j.cca.2025.120431
Molecules. 2025 Jun 17. pii: 2624. [Epub ahead of print]30(12):

Integrating NMR and MS for Improved Metabolomic Analysis: From Methodologies to Applications.

Patricia Homobono Brito de Moura, Guillaume Leleu, Grégory Da Costa, Guillaume Marti, Pierre Pétriacq, Josep Valls Fonayet, Tristan Richard.

  Metabolomics, the comprehensive analysis of low-molecular-weight metabolites (typically below 1500 DA) in biological systems, relies heavily on mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Each technique has inherent strengths and weaknesses. MS offers high sensitivity and is commonly coupled with chromatography to analyze complex matrices, yet it is destructive, has limited reproducibility, and provides limited structural information. NMR, while less sensitive, is non-destructive and enables structural elucidation and precise quantification. Recent studies increasingly employ data fusion (DF) strategies to combine the complementary information from NMR and MS, aiming to enhance metabolomic analyses. This review summarizes DF methodologies using NMR and MS data in metabolomics studies over the past decade. A comprehensive search of SciFinder, Scopus, and Clarivate Web of Science databases was conducted to analyze fusion techniques, methods, and statistical models. The review emphasizes the growing importance of DF in metabolomics, showing its capacity to provide a more comprehensive view of biochemical processes across diverse biological systems, including clinical, plant, and food matrices.

Keywords:  data fusion; mass spectrometry (MS); metabolomics; multi-omics; nuclear magnetic resonance (NMR)

DOI:  https://doi.org/10.3390/molecules30122624
Bioinformatics. 2025 Jun 17. pii: btaf354. [Epub ahead of print]

JESTR: Joint Embedding Space Technique for Ranking Candidate Molecules for the Annotation of Untargeted Metabolomics Data.

Apurva Kalia, Yan Zhou Chen, Dilip Krishnan, Soha Hassoun.

MOTIVATION: A major challenge in metabolomics is annotation: assigning molecular structures to mass spectral fragmentation patterns. Despite recent advances in molecule-to-spectra and in spectra-to- molecular fingerprint prediction (FP), annotation rates remain low.
RESULTS: We introduce in this paper a novel tool (JESTR) for annotation. Unlike prior approaches that explicitly construct molecular fingerprints or spectra, JESTR leverages the insight that molecules and their corresponding spectra are views of the same data and effectively embeds their representations in a joint space. Candidate structures are ranked based on cosine similarity between the embeddings of query spectrum and each candidate. We evaluate JESTR against mol-to-spec, spec-to-FP, and spec-mol matching annotation tools on four datasets. On average, for rank@[1-20], JESTR outperforms other tools by 55.5% - 302.6%. We further demonstrate the strong value of regularization with candidate molecules during training, boosting rank@1 performance by 5.72% across all datasets and enhancing the model's ability to discern between target and candidate molecules. When comparing JESTR's performance against that of publicly available pretrained models of SIRIUS and CFM-ID on appropriate subsets of MassSpecGym dataset, JESTR outperforms these tools by 31% and 238%, respectively. Through JESTR, we offer a novel promising avenue towards accurate annotation, therefore unlocking valuable insights into the metabolome.
AVAILABILITY: Code and dataset available at https://github.com/HassounLab/JESTR1/.
SUPPLEMENTARY INFORMATION: Available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btaf354
Life (Basel). 2025 May 22. pii: 829. [Epub ahead of print]15(6):

Steroidomics via Gas Chromatography-Mass Spectrometry (GC-MS): A Comprehensive Analytical Approach for the Detection of Inborn Errors of Metabolism.

Francesco Chiara, Sarah Allegra, Simona Liuzzi, Maria Paola Puccinelli, Giulio Mengozzi, Silvia De Francia.

  Background: Urinary steroid profiling plays a key role in the diagnosis of inherited and acquired endocrine disorders. Despite the proven diagnostic value of gas chromatography-mass spectrometry (GC-MS), standardized and clinically validated protocols for extended steroid panels remain limited. Methods: We developed and validated a GC-MS method for the quantification of 32 urinary steroid metabolites, including androgens, estrogens, progestins, glucocorticoids, and mineralocorticoids. Sample preparation involved solid-phase extraction, enzymatic hydrolysis, and dual derivatization, followed by chromatographic separation and mass detection under full scan mode. Validation followed ICH M10 guidelines. Results: The method demonstrated high selectivity, accuracy (within ±15%), and precision (CV% < 15%) across three QC levels. Limits of Quantification were estimated using the Hubaux-Vos approach and were suitable for detecting both physiological and pathological steroid concentrations. Robustness and matrix effect tests confirmed the method's reliability and reproducibility. Conclusions: This GC-MS protocol enables comprehensive urinary steroid profiling and calculation of diagnostic ratios for inborn errors of steroid metabolism and endocrine disorders. The method is suitable for clinical application and future integration into personalized medicine workflows.

Keywords:  GC-MS; inherited metabolic disorders; steroidomics; urinary steroids

DOI:  https://doi.org/10.3390/life15060829
Talanta. 2025 Jun 18. pii: S0039-9140(25)00972-5. [Epub ahead of print]296 128482

Quantitative approaches for spatial metabolomics with isomer differentiation using surface sampling capillary electrophoresis mass spectrometry.

Anastasia Golubova, Ingela Lanekoff.

  The importance of metabolites and their isomeric structures in biological function and dysfunction is increasingly recognized. However, achieving quantitative mapping of metabolites within tissue regions, particularly with isomeric specificity, remains an analytical challenge. This work presents the development of a quantitative surface sampling capillary electrophoresis method for spatial metabolomics with isomeric resolution. Five quantitation strategies were evaluated, with the optimal approach identified as sequential injection of metabolites directly from tissue alongside standards. This methodology was applied to a rat brain tissue section in a proof-of-principle study, enabling quantitative spatial analysis of metabolites, neurotransmitters, and isomeric species. Among the findings, the aromatic amino acids tyrosine, phenylalanine, and tryptophan exhibited the most dynamic distributions across four brain regions, while leucine and isoleucine demonstrated distinct spatial profiles, with leucine consistently being the more abundant isomer. This method offers a promising tool for advancing the understanding of spatially resolved biochemical processes underlying biological function and dysfunction.

Keywords:  Brain tissue section analysis; Capillary electrophoresis-mass spectrometry; Quantitative metabolomics; Spatial metabolomics

DOI:  https://doi.org/10.1016/j.talanta.2025.128482
J Pharm Biomed Anal. 2025 Jun 17. pii: S0731-7085(25)00367-X. [Epub ahead of print]265 117026

A novel UHPLC-MS/MS approach for simultaneous quantification of pyrimidine metabolites in human biofluids.

Libo Zhao, Zhini Liu, Sihan Wang, Xin Xiong, Hongzhi Zhao, Zongwei Cai, Xiaona Li.

  Pyrimidine intermediates, essential components of DNA/RNA, serve as energy transducers and signaling mediators in cellular communication pathways. However, research on pyrimidine metabolism in acute kidney injury (AKI) is hindered by the limited variety of metabolites and poorly understood underlying mechanisms. To date, no liquid chromatography coupled to mass spectrometry (LC-MS) method has achieved comprehensive coverage of pyrimidine metabolism. In this study, we developed an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous detection of 10 pyrimidine metabolites. The method demonstrated a linear range of 1.2-2400 ng/mL, with reproducible recovery rates and consistent matrix effect. Both intra-day and inter-day accuracy and precision were within acceptable limits. Most metabolites exhibited stability under various conditions, including room temperature, autosampler, -40°C preservation, and after three freeze-thaw cycles. The method enabled extensive detection of pyrimidine metabolites, ensuring effective separation of all target compounds and achieving satisfactory peak shapes, particularly for the challenging monophosphate nucleotides. The method was successfully applied to matched plasma-urine biofluids from critically ill patients. In AKI inpatients, plasma levels of uracil, thymidine, and 2'-deoxyuridine were significantly reduced, while cytosine and cytidine levels were elevated. Additionally, a decrease in urinary cytidine concentration was observed in AKI patients. These findings demonstrate disturbances in the pyrimidine metabolic pathway in AKI patients, suggesting that the onset of AKI in critically ill patients may be closely associated with the accumulation of uremic solutes. The reliable quantification provided novel insights into the disease pathogenesis of AKI.

Keywords:  Acute kidney injury (AKI); HILIC; LC-MS/MS; Pyrimidine metabolites

DOI:  https://doi.org/10.1016/j.jpba.2025.117026
Wei Sheng Yan Jiu. 2025 May;54(3): 510-519

[Determination of 27 perfluoroalkyl substances in pork by ultra-high performance liquid chromatography-tandem mass spectrometry].

Yushen Jin, Yang Xing, Kaixin Xu, Yumin Niu, Bing Shao.

   OBJECTIVE: A method based on QuEChERS and ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was established for the detection of 27 perfluoroalkyl substances(PFASs) in pork.
METHODS: Pork samples were extracted with acetonitrile-water(8∶2, V/V), and the supernatant was purified with 100 mg primary secondary amine(PSA) and 40 mg octadecylsilyl(C18). Separation was performed using an Ascentis~(TM) Express F5(2.1 mm × 150 mm, 2 μm) chromatographic column, and detection was carried out using UPLC-MS/MS with internal standard quantification.
RESULTS: The 27 PFASs exhibited good linearity in the range of 0.01-50 μg/L, with limit of detection(LOD) ranging from 0.004 to 0.044 μg/L and limit of quantification(LOQ) ranging from 0.015 to 0.145 μg/L. The intra-day and inter-day precision were determined at a spiking level of 2LOQ, with a recovery rate of 72.4%-109.7% and 78.6%-110.7%, an intra-day relative standard deviation(RSD) of 1.2%-9.6%(n=6), and an inter-day RSD of 1.0%-13.8%(n=5), respectively. Using this method, 52 pork samples from Beijing markets were tested, and the result showed that all 26 PFASs compounds, except perfluorooctanesulfonamide(PFOSA), were ubiquitous and accumulated to varying degrees in pork.
CONCLUSION: This method is simple, time-saving, and has high accuracy, sensitivity, and reliability, making it suitable for simultaneous determination of 27 PFASs in pork samples and applicable to routine monitoring and quality control of PFASs in pork samples.

Keywords:  QuEChERS; perfluoroalkyl substances; pork; ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)

DOI:  https://doi.org/10.19813/j.cnki.weishengyanjiu.2025.03.024
Biomed Chromatogr. 2025 Aug;39(8): e70155

Simultaneous Determination of Buspirone, Alprazolam, Clonazepam, Diazepam, and Lorazepam by LC-MS/MS: Evaluation of Greenness Using AGREE Tool.

Aykut Kul, Selen Al, Demet Dincel, Olcay Sagirli.

  Anxiolytic drugs such as buspirone, alprazolam, clonazepam, diazepam, and lorazepam are used to treat anxiety disorders. Measurement of drug plasma concentrations is important in the treatment of the patient. This research developed a rapid, reliable, and cost-effective LC-MS/MS method to quantify buspirone, alprazolam, clonazepam, diazepam, and lorazepam in human plasma for therapeutic drug monitoring. Furthermore, compliance with the International Council for Harmonisation (ICH) M10 Bioanalytical Method Validation and Study Sample Analysis ensured the method's reliability. Before injection into the LC-MS/MS system, the analytes and an internal standard were extracted from plasma through salt-assisted liquid-liquid microextraction (SALLME). The lower limit of quantification (LLOQ) was determined as 0.5, 2.5, 2, 30, and 10 ng/mL for buspirone, alprazolam, clonazepam, diazepam, and lorazepam, respectively. The calibration curve ranges are 0.5-50 ng/mL for buspirone, 2.5-250 ng/mL for alprazolam, 2-200 ng/mL for clonazepam, 30-3000 ng/mL for diazepam, and 10-1000 ng/mL for lorazepam, respectively, with correlation coefficients > 0.99. Its suitability for therapeutic drug monitoring was demonstrated using the method to determine drug concentration levels in real patients. The developed method has been observed to achieve a high analytical greenness metric approach and software (AGREE) score for the greenness profile.

Keywords:  LC‐MS/MS; SALLME; anxiolytic drugs; plasma; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/bmc.70155
Clin Chem Lab Med. 2025 Jun 23.

Isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedures for the quantification of total and free phenytoin in human serum and plasma.

Tobias Schierscher, Linda Salzmann, Neeraj Singh, Manuel Seitz, Janik Wild, Carina Schäfer, Friederike Bauland, Andrea Geistanger, Lorenz Risch, Christian Geletneky, Christoph Seger, Judith Taibon.

   OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of total and free phenytoin.
METHODS: Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of phenytoin from potential unknown interferences was achieved with reversed-phase chromatography, utilizing a C8 column. A protein precipitation protocol was established for preparation of total phenytoin samples, while free phenytoin samples were prepared by membrane separation utilizing a commercially available ultrafiltration device. Assay validation and determination of measurement uncertainties was performed according to the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement.
RESULTS: These RMPs demonstrated high selectivity and specificity, with no evidence of matrix effects, allowing quantification of total and free phenytoin in ranges of 0.640-48.0 μg/mL and 0.0800-4.80 μg/mL, respectively. Intermediate precision was <3.8 %, and repeatability was 1.4-3.8 %, over all concentration levels, for both forms of phenytoin. For total phenytoin, relative mean bias ranged from -2.7-0.3 % in native serum and from 0.0-1.1 % in lithium heparin plasma. Relative mean biases for free phenytoin were 3.5-4.1 % for both native serum and ultrafiltrates. Measurement uncertainties for single measurements and target value assignment were 1.8-2.5 % and 0.9-1.7 %, respectively, for total phenytoin. For free phenytoin, these measurement uncertainties were 2.0-3.9 % and 0.9-1.4 % for single measurements and target value assignment, respectively.
CONCLUSIONS: We present a novel LC-MS/MS-based RMP for phenytoin in human serum and plasma that provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Keywords:  ID-LC-MS/MS; phenytoin; qNMR; reference measurement procedure; standardization; traceability

DOI:  https://doi.org/10.1515/cclm-2024-0858
Metabolites. 2025 May 27. pii: 354. [Epub ahead of print]15(6):

SMQVP: A Web Application for Spatial Metabolomics Quality Visualization and Processing.

Zhanlong Mei, Wan Sun, Yun Zhao, Haoke Deng, Xiaolian Ning, Chunlu Feng, Jin Zi.

   BACKGROUND: Spatial metabolomics is a powerful technique that enables spatially resolved mapping of metabolite distributions at the tissue and cellular levels, providing valuable insights into biological processes. However, challenges in data quality control and preprocessing remain significant bottlenecks, critically impacting the reliability of downstream analyses and the robustness of findings.
METHODS: To address these limitations, we present Spatial Metabolomics data Quality Visualization and Processing (SMQVP v1.0), a novel software with a user-friendly graphical interface designed for the systematic quality assessment and preprocessing of spatial metabolomics data. SMQVP incorporates eight comprehensive quality visualization and evaluation modules, including background consistency assessments, noise ion filtering, intensity distribution analyses, and the identification of isotopic and adduct ions.
RESULTS: We demonstrated SMQVP's effectiveness using AFADESI-based mouse brain data, showing that the tool successfully identified and removed noise signals. This rigorous preprocessing resulted in improved clustering outcomes that more accurately reflected the underlying tissue morphology compared with analyses performed on unprocessed data.
CONCLUSIONS: SMQVP is the first systematic approach focused on quality visualization, specifically for spatial metabolomics. It offers researchers an accessible and comprehensive solution for enhancing data integrity and mitigating the impact of technical noise, thereby improving the reliability and robustness of their spatial metabolomics findings.

Keywords:  data quality control; isotopic peak detection; mass spectrometry imaging; noise ion filtering; spatial metabolomics

DOI:  https://doi.org/10.3390/metabo15060354
Anal Chem. 2025 Jun 24.

An Integrated Platform for High-Throughput Extraction and Mass Spectrometry-Based Quantification of Cholesterol and Sphingosine.

Yi-Han Lin, Yuhong Fang, Maya L Gosztyla, Edward Zhu, Abhijeet Kapoor, Andrés E Dulcey, Daniel C Talley, Shu Yang, Miao Xu, Xin Hu, Wei Zheng, Anton Simeonov, Juan J Marugan, Mark J Henderson, Christopher A LeClair, Bolormaa Baljinnyam, Dingyin Tao.

Quantification of cellular lipids in a reproducible and high-throughput manner is a key step in the development of therapeutics for lipid storage diseases. Niemann-Pick Disease Type C (NPC) is a genetic disorder characterized by the accumulation of unesterified cholesterol in late endosomes/lysosomes, which is usually measured by the filipin fluorescence assay. However, the nonspecific binding of filipin to other sterol derivatives, multiple assay steps, and difficulty in quantitation present limitations for high-throughput screening and accurate cellular cholesterol quantification. We report the development of an integrated and semiautomated protocol to extract and quantify cellular cholesterol in 384-well plates by utilizing a liquid handling platform in conjunction with a high-throughput mass spectrometry (MS) system. The 384-well plate format enables seamless lipid extraction and subsequent MS analysis in less than 2 h from a cell culture plate to final MS data. Cholesterol was extracted from neural stem cells differentiated from NPC induced pluripotent stem cells using methyl tert-butyl ether (MTBE), with 13C-cholesterol serving as an internal standard for quantification and normalization of native cholesterol. This integrated platform showed excellent quantification linearity and reproducibility (intraday and interday, R2 > 0.99) with a recovery rate between 83 and 107%. We employed this integrated platform to screen a collection of 241 investigational compounds at seven concentrations each, benchmarking the method as an efficient, label-free cellular cholesterol quantification assay for high-throughput applications. Furthermore, we demonstrated the capability to multiplex extraction and quantification of sphingosine/cholesterol in a single MS run, extending the applicability of this integrated workflow to other lipid storage diseases.

DOI: https://doi.org/10.1021/acs.analchem.4c06628
Clin Biochem. 2025 Jun 21. pii: S0009-9120(25)00089-X. [Epub ahead of print] 110960

Optimization and validation of the Kairos amino acid Kit for plasma amino acid monitoring in inherited metabolic disorder patients.

Kiara Theron, Jeannette Gauthier, Marijn Van Hulle, Murray Potter.

   PURPOSE: Genetic disorders affecting amino acid metabolism are a significant subset of inherited metabolic disorders (IMDs). Plasma amino acid (PAA) analysis is used for the diagnosis and monitoring of these disorders in order to avoid development of severe symptoms. However, PAA assays are often lengthy in analysis time (>2h/sample) and some methods lack specificity and sensitivity. This project offers a novel solution through the optimization and clinical validation of the Kairos Amino Acid Kit by Waters Corp.
DESIGN AND METHODS: Using liquid chromatography with tandem mass spectrometry and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization, amino acid quantification has been achieved for 45 amino acids in 15 min of chromatography time per sample. The method utilizes reversed-phase chromatography via a high-strength silica C18 column. The optimized column chemistry enables strong hydrophobic retention, resolving all isobaric amino acids for individual mass-spectrometer quantification. Method validation protocols include linearity, sensitivity, precision, and bias.
RESULTS: Clinical validation has been performed, indicating high reproducibility and clinical applicability. Linearity results demonstrated 42/45 amino acids with R2 > 0.975 over a linear range of at least 5-1000 µmol/L. Spiked plasma calibrators demonstrated high recovery with R2 > 0.971. Twenty-day precision testing demonstrated total coefficient of variation < 15 % and sensitivity testing confirmed all analytes have limits of detection < 5 µmol/L, indicating high analytical sensitivity and precision. Method comparison to Waters' MassTrak Kit demonstrates acceptable bias and supports a future study to update reference intervals.
CONCLUSION: The clinical validation of the Kairos Amino Acid Kit for plasma amino acid monitoring highlights the potential of this novel method to enhance amino acid quantification in clinical laboratories for IMD management.

Keywords:  Amino acid monitoring; Clinical validation; Derivatization; IMD; LC-MS/MS; Optimization

DOI:  https://doi.org/10.1016/j.clinbiochem.2025.110960
J Mass Spectrom Adv Clin Lab. 2025 Aug;37 39-48

Development of an LC-MS/MS method for quantification of colistin and colistin methanesulfonate in human plasma and its application to stability studies and therapeutic drug monitoring.

Tinghui Zhao, Lu Liu, Guangjie Yang, Hengyi Yu, Lihui Qiu, Xiping Li, Dong Xiang, Xuepeng Gong.

   Introduction: Colistin serves as the last line of defense against multidrug-resistant Gram-negative bacterial infections and is commonly administered in clinical practice as its prodrug, colistin methanesulfonate (CMS). However, due to its notable nephrotoxicity and narrow therapeutic window, therapeutic drug monitoring (TDM) is essential.
Objectives: To develop an optimal LC-MS/MS method for the quantification of colistin and CMS in human plasma and to apply it to stability studies and TDM.
Methods: Colistin A, colistin B, and internal standard (IS, polymyxin B2) were extracted from plasma using solid phase extraction columns. Sample separation was performed using a Welch Ultimate LP-C18 column with a 5-minute gradient elution consisting of water and acetonitrile, both supplied with 1.0% formic acid. The CMS concentration was obtained by comparing the total amount of colistin in acid-hydrolyzed and non-acid-hydrolyzed plasma.
Results: Colistin A and colistin B showed excellent linearity in the concentration range of 0.1-10.0 μg/mL (R2 > 0.995) with acceptable specificity, accuracy (90.97 %-114.65 %), precision (RSD < 15 %), matrix effect (RSD < 15 %), and recovery (91.93 %-100.93 %). CMS in five commonly used clinical infusion solutions was stable when stored at room temperature for 8 h or at 4 °C for 24 h. The whole blood and plasma samples of CMS are susceptible to degradation at room temperature but are stable on ice. Plasma concentrations of colistin and CMS were accurately determined in three critically ill patients.
Conclusion: The method we have developed is robust and streamlined, and has successfully demonstrated the potential feasibility for future TDM applications of colistin and CMS in critically ill patients.

Keywords:  Colistin; Colistin methanesulfonate; Liquid chromatography-tandem mass spectrometry; Pharmacokinetics; Stability

DOI:  https://doi.org/10.1016/j.jmsacl.2025.05.001
Food Chem Toxicol. 2025 Jun 25. pii: S0278-6915(25)00374-6. [Epub ahead of print] 115606

Simultaneous Biomonitoring of Common Food Carcinogen Acrylamide and its Major Metabolites using Fast Urinary Metabolites Extraction Technique Coupled with LC-MS/MS Analysis.

Pei-Chien Tsai, Karthikeyan Prakasham, Swapnil Gurrani, Shih-Tao Hu, Po-Chin Huang, Jentaie Shiea, Vimalkumar Krishnamoorthi, Ming-Tsang Wu, Chia-Fang Wu, Vinoth Kumar Ponnusamy.

  Acrylamide (AA) is a probable carcinogen, often found in the environment and food, which causes various cancers in humans. Therefore, it is vital to have a facile, quick, sensitive analytical method for biomonitoring AA and its metabolites (including glycidamide (GA), N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA)) in human urine sample for exposure risk and toxicology assessment applications. In this study, we presented a rapid sample pre-treatment technique named "fast urinary metabolites extraction technique" (FaUMEx) coupled with UHPLC-MS/MS method for efficient biomonitoring of AA and its metabolites in human urine samples. In the FaUMEx technique, two syringes were integrated for liquid-liquid extraction, followed by a micro-solid phase clean-up process. The fully optimized and validated FaUMEx/UHPLC-MS/MS technique yielded good detection and quantification levels (LOD of 0.01 to 0.2 ng/mL, and LOQ of 0.5 to 0.05 ng/mL for AA, GA, AAMA, and GAMA, respectively). The inter- and intra-day analysis shows reliable relative recoveries ranging from 82.90-116.80% with <12% RSD. Moreover, the matrix effect ranged from -8.9 % to +7.7%, demonstrating the excellent cleanup efficiency towards quantifying AA and its metabolites from the human urine samples with minimal matrix interferences. Thus, the presented method is simple, efficient, and sensitive, and can be applied for biomonitoring applications.

Keywords:  Acrylamide and its metabolites; In-syringe based fast urinary metabolites extraction technique; UHPLC-MS/MS; biomonitoring; human urine

DOI:  https://doi.org/10.1016/j.fct.2025.115606
J Am Soc Mass Spectrom. 2025 Jun 24.

A Compact Method Specialized for Full Analysis of the Glycan Composition of Jellyfish Mucin Using Limited Degradation Followed by High-Resolution Mass Spectrometry.

Shiori Kaise, Minami Sugiyama, Shinra Tanaka, Takuma Kaneko, Hiroshi Inui, Kiminori Ushida.

  A compact and specialized method for analyzing the O-glycan composition of jellyfish mucin (Q-mucin) has been newly developed. Q-mucin was analyzed using high-resolution mass spectrometry (HRMS) with electrospray ionization (ESI), combined with limited degradation (LD) by endoprotease Glu-C. Direct analysis of the solution after enzymolysis caused unwanted dissociation of glycan adducts from tandem repeats (TRs). However, the introduction of HPLC prior to ESI, utilizing a reverse-phase column with an aqueous solution of formic acid and acetonitrile as the mobile phase, successfully minimized the source dissociation of many glycan adducts from TRs. As a result, we established a rapid and cost-effective protocol for analyzing the O-glycans in Q-mucin over a wide dynamic range. Q-mucin contains two types of TRs that are commonly shared by a large number of jellyfish species. However, the residual challenge lies in understanding the O-glycan composition, which varies depending on species and ecological factors. For systematic studies across a vast number of species, the newly developed protocol, which does not rely on complex analytical algorithms, proves highly suitable. Given its superior time efficiency and accuracy compared to large-scale mass analysis programs designed for general use, we propose that the development of purpose-oriented methods holds significant value for specialized studies using mass spectrometry.

Keywords:  electrospray ionization; glycoform analysis; glycoprotein; jellyfish mucin; limited degradation

DOI:  https://doi.org/10.1021/jasms.5c00016
Animals (Basel). 2025 Jun 06. pii: 1687. [Epub ahead of print]15(12):

Establishment and Validation of Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Aldosterone Quantification in Feline Serum with Reference Interval Determination.

Tommaso Furlanello, Francesca Maria Bertolini, Andrea Zoia, Jose Sanchez Del Pulgar, Riccardo Masti.

  Aldosterone, a mineralocorticoid hormone synthesised in the adrenal cortex, is essential for maintaining electrolyte balance and fluid homeostasis. Its role in feline physiology remains underexplored, despite its importance in regulating sodium reabsorption and potassium excretion via mineralocorticoid receptors in renal tubules. This study is warranted given aldosterone's importance in cats, particularly in light of their unique physiological traits, including highly concentrated urine and sensitivity to hydration status. Primary hyperaldosteronism, the most common feline adrenocortical disorder, contributes to arterial hypertension and chronic kidney disease, yet often remains underdiagnosed due to overlapping symptoms like hypokalaemia and hypertension. This research aimed to validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure serum aldosterone and to establish a reference interval in a population of healthy cats across a broad age range. The method demonstrated high precision and accuracy, with inter-assay coefficients of variation under 15%. Analysis of 49 healthy cats (40 young, 9 old) revealed a reference interval of 5.0-78.4 pg/mL (13.8-217.2 pmol/L). These findings provide a robust framework for diagnosing aldosterone-related disorders in cats and underscore the need for species-specific diagnostic tools. Improved understanding of aldosterone's role could refine treatment strategies and enhance outcomes for affected feline patients.

Keywords:  LC-MS/MS; adrenal gland; cat; mineralocorticoid hormone

DOI:  https://doi.org/10.3390/ani15121687
J Pharm Biomed Anal. 2025 Jun 18. pii: S0731-7085(25)00377-2. [Epub ahead of print]265 117036

Laser capture microdissection-assisted gas chromatography-triple-quadruple mass spectrometry for spatial metabolic profiling of esophageal squamous cell carcinoma.

Peng Dou, Huan Yang, Miao Bao, Yanli Li, Xinyu Liu, Xinxin Liang, Xin Lu, Jinhu Fan, Guowang Xu.

  With the development of precision medicine and single-cell analysis, spatial metabolomics has become a hotspot in the biomedical field. However, current spatial metabolomics methods are still unable to meet the demand for the integration of spatial omics in terms of metabolite coverage, isomer differentiation, and applicability of clinical samples. Therefore, this study developed an analytical strategy based on laser capture microdissection-assisted gas chromatography-triple-quadruple mass spectrometry to characterize metabolic profile differences in clinical tissue sections. The extraction and derivatization methods were optimized separately. Analytical characteristics evaluation showed intra- and inter-day precision within 20 %, with most metabolites exhibiting linear ranges spanning three orders of magnitude. Using this method, metabolic profiles of five morphologies in esophageal squamous cell carcinoma (ESCC) and adjacent normal tissue (ANT) sections were characterized, identifying 88 metabolites across multiple categories. ANT tissues exhibited metabolic diversity across different morphologies, with cancer nest metabolic profiles in ANT being closer to those of ESCC. Squamous epithelial hyperplasia tissues showed dramatically lower levels of amino-containing compounds than other morphologies. This study presents a spatial metabolomics analysis method with good robustness and clinical applicability, offering valuable methodological support for clinical spatial omics applications.

Keywords:  Esophageal squamous cell carcinoma; Gas chromatography-tandem mass spectrometry; Laser capture microdissection; Spatial metabolomics

DOI:  https://doi.org/10.1016/j.jpba.2025.117036
Metabolomics. 2025 Jun 22. 21(4): 86

Introducing Korea metabolomics data repository (KMAP): bridging Korean metabolomics data to global data sharing infrastructure.

Metabolomics Data Curation Center (MDCC)

  The Korea MetAbolomics data rePository (KMAP), available at https://kbds.re.kr/KMAP , is a public repository for metabolomics datasets developed as a part of the Korea BioData Station (K-BDS). KMAP archives metabolomics data and metadata generated from government-funded research projects in Korea, regardless of sample origin or analytical techniques. While data collection is nationally coordinated, data sharing is intended to be global. Here, we present our recent efforts to align KMAP with international standards for QA/QC and interoperability with other repositories.

Keywords:  Data curation; Data repository; FAIR; Korea metabolomics data repository; Quality control

DOI:  https://doi.org/10.1007/s11306-025-02285-5
Environ Health (Wash). 2025 Jun 20. 3(6): 596-604

Breath and Blood Metabolomics: A Comparative Study Using SESI-HRMS/MS and UHPLC-ESI-HRMS/MS.

Zhifeng Tang, Jianming Yang, Xin Xu, Keda Zhang, Huiling Wang, Xin Luo, Mingliang Fang, Tao Huan, Xue Li.

  Breath metabolomics enables noninvasive and rapid acquisition of metabolic information by detecting volatile organic compounds (VOCs) in exhaled breath. Secondary electrospray ionization high-resolution tandem mass spectrometry (SESI-HRMS/MS) offers the highest coverage for detecting breath metabolites among current real-time breath analysis techniques. Although it has been generally recognized that metabolites in breath originate from the blood, a molecular-level understanding of the characteristics of metabolites in both breath and blood remains insufficient. In this study, nontargeted analyses of breath and blood samples from 11 healthy volunteers were performed using SESI-HRMS/MS and ultrahigh performance liquid chromatography electrospray ionization high-resolution tandem mass spectrometry (UHPLC-ESI-HRMS/MS), respectively. Tandem mass spectrometry was employed for metabolite annotation. Twenty-six breath-unique metabolites and 73 blood-unique metabolites were identified. Besides, seven metabolites were found in both breath and blood, including 7-oxabicyclo [2.2.1] heptane, levulinic acid, indole, pyroglutamic acid, malic acid, glutamic acid, and histidine. Intriguingly, the correlation of these metabolites between breath and blood was low (r < 0.4 or p > 0.05). Among all the confirmed metabolites, breath metabolites exhibit higher volatility according to their water-gas partition coefficient (log P w/g) compared to blood metabolites. In addition, gender-derived differences in breath were significantly smaller than blood. In summary, this study indicates that breath metabolites are likely to offer complementary information on blood metabolites. When combined with blood metabolomics, this would be advantageous for the appropriate application of breath metabolomics in life sciences, such as in biomarker discovery.

Keywords:  SESI-HRMS/MS; UHPLC-ESI-HRMS/MS; blood metabolomics; breath metabolomics; metabolite identification; metabolomics comparison

DOI:  https://doi.org/10.1021/envhealth.4c00248
Metabolites. 2025 Jun 17. pii: 407. [Epub ahead of print]15(6):

An LC-MS Method to Quantify Rhein and Its Metabolites in Plasma: Application to a Pharmacokinetic Study in Rats.

Nyma Siddiqui, Yuan Chen, Ting Du, Yang Wang, Charmeyce Buck, Song Gao.

  Background: Diacerein, a prodrug of Rhein, is commonly prescribed for the management of joint disorders, specifically osteoarthritis. This study aimed to develop and validate an LC-MS/MS method to quantify Rhein and its major metabolites, Rhein-G1 and Rhein-G2, in plasma samples. Method: An ACE C18 column was used for chromatographic separation with a mobile phase comprising ammonium acetate at a concentration of 1.0 mM and acetonitrile. Detection was achieved using a Sciex 4000 Q-Trap LC-MS/MS, operated in negative ion mode with multiple reaction monitoring (MRM). Results: The analytical results indicated that the lower limit of quantification (LLOQ) for Rhein and its glucuronides was 7.81 nM. Precision was consistently below 9.14%, while accuracy remained within the acceptable range of 80.1-104.2%. We also verified the method's matrix effect recovery and stability variance, which were less than 12.60% and 10.37%, respectively. The pharmacokinetic study demonstrated that diacerein is swiftly metabolized into Rhein, and then Rhein subsequently undergoes glucuronidation, forming detectable concentrations of Rhein-G1 and Rhein-G2 in plasma. Conclusions: This new LC-MS/MS method proved to be both sensitive and selective, allowing for pharmacokinetic studies in rats.

Keywords:  LC-MS; PK; Rhein; Rhein-glucuronides; diacerein

DOI:  https://doi.org/10.3390/metabo15060407
Food Chem. 2025 Jun 24. pii: S0308-8146(25)02548-8. [Epub ahead of print]491 145297

Quantitative analysis of seven free short-chain fatty acids in infant formula using GC-MS/MS.

Yunxiao Zhu, Yang Liu, Qiu Sun, Jia Chen, Mengling Tu, Yehua Han, Xianjiang Li.

  Free short-chain fatty acids (FSCFAs) are recognized as essential nutrients in infant formula. This study aims to develop a rapid, sensitive, and efficient method for analyzing seven FSCFAs in infant formula. The sample preparation process utilized an acetonitrile-anhydrous magnesium sulfate system for the FSCFAs extraction. The enriched FSCFAs were derivatized using butanol-acetyl chloride to enhance stability and chromatographic separation. Analysis of the resulting fatty acid butyl esters (FABEs) was performed using gas chromatography-tandem mass spectrometry (GC-MS/MS). Under optimized conditions, the chromatographic method achieved baseline separation of seven FABEs within 14.8 min, demonstrating excellent linearity and reproducibility. The limits of detection and quantification ranged from 12.0 to 34.9 ng/g and 40.0 to 116.5 ng/g, respectively. In the tested real samples, six FSCFAs were successfully detected. These findings provide a scientific basis for optimizing the nutritional composition of infant formula and improving its similarity to human milk.

Keywords:  Fatty acid butyl esters; Free short-chain fatty acids; GC–MS/MS; Infant formula

DOI:  https://doi.org/10.1016/j.foodchem.2025.145297
Anal Bioanal Chem. 2025 Jun 27.

High-throughput quantification of butachlor by DART-MS/MS coupled with micro-solid-phase extraction and dual fragmentation to enhance selectivity and sensitivity.

Huaqiang Li, Feifei Wu, Yuxian Ding, Guozhou Long, Lei Yin.

  A novel and rapid analytical assay based on direct analysis in real-time tandem mass spectrometry (DART-MS/MS) technology was established for quantification of butachlor in this study. The assay incorporated micro-solid-phase extraction (μ-SPE) for sample pretreatment, with acetochlor as an internal standard. Quantification was achieved via transitions from [M + H]+ precursor ions to primary and secondary fragment ions at m/z 312.1 → 238.0 → 162.1 (butachlor) and 270.1 → 224.1 → 148.0 (acetochlor). This chromatography-free approach eliminated reliance on mobile phases and chromatographic instrumentation, significantly improving analytical throughput (0.4 min/sample) while reducing environmental impact. Method validation demonstrated linearity over 0.5-100 ng/mL (R2 > 0.995), with intra-/inter-day precision < 13.1% and accuracy ranging from -6.00% to 11.0%. The assay was used to evaluate the cellular uptake kinetics of butachlor in MCF-7 cells. These findings underscore the utility of DART-MS/MS assay for rapid environmental and toxicological assessments while highlighting potential safety concerns associated with butachlor exposure. The integration of DART-MS/MS with μ-SPE offers a sustainable alternative to conventional bioanalytical workflows, combining speed, sensitivity, and reduced chemical consumption for ecotoxicological studies.

Keywords:  Butachlor; Cellular uptake; DART-MS/MS; Micro-solid-phase extraction; Quantification

DOI:  https://doi.org/10.1007/s00216-025-05975-0