bims-metlip 2025-04-06 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–04–06
28 papers selected by
Sofia Costa, Matterworks

HeuSMA: A Multigradient LC-MS Strategy for Improving Peak Identification in Untargeted Metabolomics.
A comparative study of data-independent acquisition and data-dependent acquisition in liquid chromatography-mass spectrometry-based untargeted metabolomics analysis of Panax genus sample.
Development and Validation of a Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantitation of Vitamin K Metabolites in Different Matrices.
Advances in Tandem Mass Spectrometry Imaging for Next-Generation Spatial Metabolomics.
A Rapid and Sensitive Method for the Determination of Bisoprolol in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry.
Optimization and validation of a multi-residue method for analyzing organic UV absorbers in human urine by UHPLC-MS/MS.
Development and validation of an LC-MS/MS method with precolumn derivatization for the determination of dimethoxyethyl phthalate metabolites ethylene glycol methyl ether and methoxyacetic acid in rat plasma.
Implementation and validation of a 24/7 system for the monitoring of antiepileptic drugs.
A novel ultra-sensitive LC-MS/MS method for determination of elobixibat in human plasma; Application to a bioequivalence study on healthy volunteers.
OzMALDI: A Gas-Phase, In-Source Ozonolysis Reaction for Efficient Double-Bond Assignment in Mass Spectrometry Imaging with Matrix-Assisted Laser Desorption/Ionization.
Rapid Determination of 15 Herbicides in Blood and Soil by Direct Analysis in Real Time-Tandem Mass Spectrometry.
Norm ISWSVR Enhanced Data Repeatability and Accuracy in Large-Scale Targeted Quantification Metabolomics.
An LC-MS/MS Multiplexed Method to Quantify 14 Sphingolipids in Human Cerebrospinal Fluid (CSF).
Pentafluorobenzyl bromide - A versatile derivatization agent in chromatography and mass spectrometry: II. Analysis of organic acids and bases, and comparison with other perfluorinated reagents.
Elucidation of the Human Cerebrospinal Fluid Metabolome by Ion Mobility Mass Spectrometry and NMR Spectroscopy.
Establishment of the isotope dilution-gas chromatography-tandem mass spectrometry (ID-GC/MS) method for accurate quantification of phenylalanine and its application to value assignment of external quality assessment samples.
Rapid MALDI-MS/MS-Based Profiling of Lipid A Species from Gram-Negative Bacteria Utilizing Trapped Ion Mobility Spectrometry and mzmine.
Optimizing Mirabegron Stability in Human Plasma: Method Development, Validation Through Integration of Esterase Inhibitors and Stabilizers.
Mass Spectrometry Applied to Human Cerebrospinal Fluid Lipidome (II).
Conversion Reaction of Stable-Isotope Oxygen Labeling of Carboxylic Acids for Accurate Screening LC-MS/MS Assay: Application of Behavioral Changes of Short-Chain Fatty Acids in Sports Athletes under Exercise Loading.
Denoising Search doubles the number of metabolite and exposome annotations in human plasma using an Orbitrap Astral mass spectrometer.
Qualitative and quantitative lipidomic profiling of cardiolipin, oxidized cardiolipin, and monolysocardiolipin from human skeletal muscle by UPLC-Orbitrap-MSn.
Synthetic cannabinoids receptor agonists in oral fluid: development of a dispersive liquid-liquid microextraction (DLLME) method with liquid chromatography-mass spectrometry detection.
Data-Independent Acquisition Coupled with Electron-Activated Dissociation for In-Depth Structure Elucidation of the Fatty Acid Ester of Hydroxy Fatty Acids.
metLinkR: Facilitating Metaanalysis of Human Metabolomics Data through Automated Linking of Metabolite Identifiers.
Evaluating Ion Mobility Data Acquisition, Calibration, and Processing for Small Molecules: A Cross-Platform Assessment of Drift Tube and Traveling Wave Methodologies.
Development and Validation of a Sensitive LC-MS/MS Method for Simultaneous Determination of Several Nitrosamines in Large Volume Parenteral.
3D Mass Spectrometry Imaging as a Novel Screening Method for Evaluating Biocontrol Agents.

Anal Chem. 2025 Apr 03.

HeuSMA: A Multigradient LC-MS Strategy for Improving Peak Identification in Untargeted Metabolomics.

Yao-Yu Chen, Na An, Yan-Zhen Wang, Peng-Cheng Mei, Jun-Di Hao, Song-Mei Liu, Quan-Fei Zhu, Yu-Qi Feng.

Metabolomics, which involves the comprehensive analysis of small molecules within biological systems, plays a crucial role in elucidating the biochemical underpinnings of physiological processes and disease conditions. However, current coverage of the metabolome remains limited. In this study, we present a heuristic strategy for untargeted metabolomics analysis (HeuSMA) based on multiple chromatographic gradients to enhance the metabolome coverage in untargeted metabolomics. This strategy involves performing LC-MS analysis under multiple gradient conditions on a given sample (e.g., a pooled sample or a quality control sample) to obtain a comprehensive metabolomics data set, followed by constructing a heuristic peak list using a retention index system. Guided by this list, heuristic peak picking in quantitative metabolomics data is achieved. The benchmarking and validation results demonstrate that HeuSMA outperforms existing tools (such as MS-DIAL and MZmine) in terms of metabolite coverage and peak identification accuracy. Additionally, HeuSMA improves the accessibility of MS/MS data, thereby facilitating the metabolite annotation. The effectiveness of the HeuSMA strategy was further demonstrated through its application in serum metabolomics analysis of human hepatocellular carcinoma (HCC). To facilitate the adoption of the HeuSMA strategy, we also developed two user-friendly graphical interface software solutions (HPLG and HP), which automate the analysis process, enabling researchers to efficiently manage data and derive meaningful conclusions (https://github.com/Lacterd/HeuSMA).

DOI: https://doi.org/10.1021/acs.analchem.4c05315
Anal Bioanal Chem. 2025 Apr 02.

A comparative study of data-independent acquisition and data-dependent acquisition in liquid chromatography-mass spectrometry-based untargeted metabolomics analysis of Panax genus sample.

Yi Wu, Yang Wang.

  Data-independent acquisition (DIA) and data-dependent acquisition (DDA) are frequently employed in the execution of tandem mass spectrometry (MS2) analyses. This study explored the application of DIA (MSe) and DDA (fast-DDA) in liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics using Panax genus samples. MSe provided comprehensive sample information, extracting more ion peaks with better peak shape and increased scan points compared to fast-DDA. Features from MSe data are four times more than those from fast-DDA data. Fast-DDA, however, delivered high-quality MS2 data, enhancing compound annotation via the GNPS web tool. Database matches with fast-DDA data were nearly 35 times greater than those using MSe data. Therefore, combining MSe and fast-DDA can improve data analysis and metabolite annotation. An improved workflow integrating DIA and DDA was proposed, requiring additional QC sample injections for DDA analysis but eliminating the need for sample reprocessing and re-analysis, thus saving time and resources. The established workflow was applied to the Panax genus samples analysis to confirm its applicability. This study offers a deeper understanding of DIA and DDA, guiding the selection of data acquisition strategies for LC-MS-based untargeted metabolomics.

Keywords:   Panax genus; GNPS; Liquid chromatography-mass spectrometry; Molecular networking; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s00216-025-05861-9
J Mass Spectrom. 2025 Apr;60(4): e5120

Development and Validation of a Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantitation of Vitamin K Metabolites in Different Matrices.

Danchen Wang, Yichen Ma, Honglei Li, Xiaoli Ma, Yutong Zou, Songlin Yu, Ling Qiu.

  Adequate vitamin K is crucial for optimal health. Although vitamin K detection methods have been established using liquid chromatography-tandem mass spectrometry (LC-MS/MS), some limitations remain. Therefore, we aimed to establish a stable and rapid LC-MS/MS method that can quantify phylloquinone (VK1), menaquinones-4 (MK-4), and menaquinones-7 (MK-7) in serum and cerebrospinal fluid and explore its clinical applications. We developed an LC-MS/MS method with atmospheric pressure chemical ionization to quantify and validate its performance according to Clinical Laboratory and Standard Institution standards (C62-Ed2). Serums from 50 healthy individuals and cerebrospinal fluid from 15 patients were collected for clinical application. Sample preparation involved lipase incubation, protein precipitation with ethanol, and liquid-liquid extraction with hexane/ethyl; optimization was performed for sample preparation and LC separation. Linearity was 50-10 000 pg/mL for VK1, MK-4, and MK-7. The total coefficient of variation (%) for VK1, MK-4, and MK-7 ranged from 8.5% to 10.4%, 8.0% to 10.4%, and 7.0% to 11.1%, respectively. Recovery of VK1, MK-4, and MK-7 was 82.3%-110.6%, 92.3%-110.6%, and 89.5%-117.8%, respectively. VK1 and MK-7 were detected in the serum of all 50 healthy subjects, whereas MK-4 was detected in only 13 (26%) subjects. Approximately 53.3% (8/15) had no detectable vitamin K in their cerebrospinal fluid. The developed method exhibited satisfactory performance and was applicable for detecting VK1, MK-4, and MK-7 in serum and cerebrospinal fluid.

Keywords:  fat‐soluble vitamin; liquid chromatography tandem–mass spectrometry; menaquinones‐4; menaquinones‐7; vitamin K

DOI:  https://doi.org/10.1002/jms.5120
Anal Chem. 2025 Apr 02.

Advances in Tandem Mass Spectrometry Imaging for Next-Generation Spatial Metabolomics.

Yao Qian, Xiaoxiao Ma.

Spatial metabolomics based on mass spectrometry imaging (MSI) is a promising approach for fundamental biological research and disease biomarker discovery. It simultaneously reveals the spatial distributions of hundreds of metabolites across tissue sections. While previous MSI experiments predominantly rely on high-resolution mass analysis for metabolite annotation, the high specificity in resolving molecular structures is essential to distinguish isomers or isobars to obtain ultimate identities of the metabolites. This is also critical for correlating their biological functions with spatial distribution patterns. Tandem mass spectrometry (MS/MS) is effectively used to obtain molecular structural information and has been integrated into MSI for spatial mapping of structurally distinct biomolecules, though typically with low coverage. The main technical challenge in achieving high-coverage, high-structure-resolving spatial mapping of biomolecules lies in the limited amount of sample available from each tissue pixel in conventional MS/MS analysis, which restricts the number of MS/MS scans that can be conducted on the metabolite precursors of interest. In this Perspective, we highlight recent developments in advanced MS/MS imaging strategies aimed at achieving high-coverage spatial metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.5c00157
Biomed Chromatogr. 2025 May;39(5): e70054

A Rapid and Sensitive Method for the Determination of Bisoprolol in Human Plasma by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry.

Yihong Jiang, Yaling Niu, Congchao Ning, Feng Qin, Chengda Yan, Xiumei Lu.

  A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of bisoprolol in human plasma was established and validated. The sample was pretreated by methanol precipitation protein, and the isotope bisoprolol-d5 was used as the internal standard. The chromatographic column was ACQUITY UPLC BEH-C18 column (2.1 × 50 mm, 1.7 μm), with methanol and 0.2% formic acid aqueous solution as mobile phase for gradient elution. The electrospray ionization (ESI) source was used in the positive ion mode, and the multiple reaction monitoring (MRM) mode was used. The total running time was only 2.00 min. The correlation coefficient was good (r > 0.99) in the linear range of 0.0200-40.0 ng/mL. The lower limit of quantitation (LLOQ) was 20.0 pg/mL. The intrabatch and interbatch precisions were not more than 8.9% and 9.2%. The intrabatch and interbatch accuracies were -7.9% ~ 6.3% and -6.9% ~ 5.0%. The method was fully validated including whole blood stability and reinjection reproducibility and successfully applied to the pharmacokinetic study of 5-mg bisoprolol in healthy volunteers, which 93.1% incurred samples reanalysis (ISR) met the criteria. Compared with the reported methods, this method had the highest sensitivity and fast analysis speed.

Keywords:  UHPLC‐MS/MS; bisoprolol; pharmacokinetics; plasma

DOI:  https://doi.org/10.1002/bmc.70054
Talanta. 2025 Mar 26. pii: S0039-9140(25)00521-1. [Epub ahead of print]293 128031

Optimization and validation of a multi-residue method for analyzing organic UV absorbers in human urine by UHPLC-MS/MS.

Xiao Lin, Dejun Bao, Qi Sun, Zhuangzhuang Feng, Xiaojian Hu, Xu Zhang, Ying Zhu.

  Organic UV absorbers (OUVAs) have gained increasing public concern over the past several years due to their potential adverse effects on humans. The currently available methods for determining OUVAs in humans are typically designed to detect only a limited number of these compounds and lack sufficient sensitivity. In this study, we established a simple and sensitive analytical method that combines liquid-liquid extraction (LLE) with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the determination of 24 OUVAs and metabolites in human urine samples. This method efficiently analyzes a variety of OUVAs, including benzophenones, benzotriazoles, cinnamates, salicylates, camphor derivatives, triazines, dibenzoylmethane, and aminobenzoic acid derivative. Two extraction approaches, solid-phase extraction (SPE) and LLE, along with various extraction solvents and extraction times were investigated to optimize sample treatment with the aim of achieving high recoveries and minimizing matrix effects. The recoveries of 24 target analytes at three spiked levels (0.02, 0.5 and 2.5 ng/mL) ranged from 70.4 % to 130 %. The lower limits of quantification were 0.004 ng/mL to 0.028 ng/mL for benzophenones, 0.005 ng/mL to 0.012 ng/mL for benzotriazoles, 0.004 ng/mL to 0.026 ng/mL for cinnamates, 0.013 ng/mL to 0.031 ng/mL for salicylates, 0.003 ng/mL to 0.009 ng/mL for camphor derivatives, 0.01 ng/mL to 0.015 ng/mL for triazines, 0.006 ng/mL for dibenzoylmethane, and 0.017 ng/mL for aminobenzoic acid derivative. This is the first study to present a valuable method for the simultaneous determination of multiple urinary OUVAs using a single preprocessing method and dual injection. The analytical method was used for the analysis of OUVAs in 48 urine samples collected from healthy individuals. Eighteen OUVAs were detected, with detection rates ranging from 2.08 % to 100 %, indicating widespread exposure to these compounds among the Chinese population.

Keywords:  Exposure assessment; Human urine; Liquid-liquid extraction; Organic UV absorbers; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.talanta.2025.128031
J Pharm Biomed Anal. 2025 Mar 28. pii: S0731-7085(25)00188-8. [Epub ahead of print]261 116847

Development and validation of an LC-MS/MS method with precolumn derivatization for the determination of dimethoxyethyl phthalate metabolites ethylene glycol methyl ether and methoxyacetic acid in rat plasma.

Xinran Cui, Yongbin Wang, Yan Zhan, Xiaoyan Chen.

  Dimethoxyethyl phthalate (DMEP) is an industrial phthalates reagent exposed to human body. Its hydrolytic metabolite ethylene glycol methyl ether (EGME) and further oxidative metabolite methoxyacetic acid (MAA) have reproductive and developmental toxicity, and the latter is more toxic than the former. However, due to the lack of sensitive and reliable analytical method, the in vivo clearance and biotransformation of EGME and MAA is poorly understood. In order to evaluate their toxicokinetic profiles, a precolumn derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of EGME and MAA in rat plasma. After a simple protein precipitation procedure, the sample was derivatized with p-toluene sulfonyl isocyanate (PTSI) and 4-bromo-N-methylbenzylamine (4-BNMA), separated by gradient elution on HSS T3 column, and detected by electrospray ionization (ESI) source in positive multiple reaction monitoring (MRM) mode. The results of the method validation showed that EGME had good linearity in the range of 2.00-400 ng/mL and MAA in the range of 5.00-1000 ng/mL. The intra- and inter-day accuracy and precision were within acceptable limits ( ± 15 %) at all concentrations. This validated method was successfully applied to characterize the toxicokinetics of EGME and MAA in rats following a gavage administration of 50 mg/kg DMEP.

Keywords:  Derivatization; Dimethoxyethyl phthalate; Ethylene glycol methyl ether; LC-MS/MS; Methoxyacetic acid

DOI:  https://doi.org/10.1016/j.jpba.2025.116847
Front Neurol. 2025 ;16 1493201

Implementation and validation of a 24/7 system for the monitoring of antiepileptic drugs.

Tatjana Khromov, Gry Helene Dihazi, Phillipp Brockmeyer, Andreas Fischer, Frank Streit.

   Background: Epilepsy is a common neurological disorder associated with seizures that impact patients' quality of life. Treatment includes antiepileptic drugs (AEDs), each effective only at a specific dose, making continuous therapeutic drug monitoring (TDM) useful in clinical cases under inpatient conditions. Conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) lacks automation for 24/7 operation, limiting clinical applicability. This study validates a fully automated 24/7 AED monitoring system using the Clinical Laboratory Automated Sample Preparation Module 2030 (CLAM-2030).
Methods: The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines by evaluating linearity, precision, accuracy, carry over, matrix effects, and calibration stability. Twenty-six AEDs were quantified in plasma using multiple reaction monitoring (MRM) transitions in positive and negative electrospray ionization modes. Sample preparation was fully automated: 20 μL methanol was used to wet the column, followed by 20 μL internal standard and 100 μL acetonitrile for protein precipitation. The supernatant was filtered and injected directly into the LC system. Chromatographic separation was achieved within 4.5 min using a C18 column (2.1 × 50 mm, 2.7 μm) under gradient conditions with a mobile phase of 0.2 mM ammonium formate and 0.002% formic acid.
Results: The method demonstrated excellent linearity over the validated concentration ranges (R 2 > 0.99 for all analytes). Within-run imprecision was <15% at the lower limit of quantitation (LLOQ), while between-run imprecision was <10% for most AEDs. Accuracy was within ±10% of nominal concentrations at all quality control (QC) levels. Matrix effects were within acceptable limits (<30% variation) for 23 of 26 analytes, with compensatory corrections applied for carbamazepine-D10, felbamate-D4, and levetiracetam-D6. Carry over was negligible [<2% for all AEDs except retigabine and N-desmethylselegiline (NDMS), which remained below 6.5%]. Calibration stability was maintained over 5 days with concentration and peak area variation <10%. An interlaboratory comparison (ring test) showed a relative standard deviation <20% for all analytes.
Conclusion: This study establishes a robust, fully automated, high-throughput method for continuous AED monitoring in the clinical setting. The CLAM-2030-LCMS-8060NX system enables reliable 24/7 TDM with minimal technical expertise, ensuring optimized AED therapy and improved patient outcomes.

Keywords:  24/7 automation; CLAM-2030; LC-MS/MS; antiepileptic drugs; epilepsy; therapeutic drug monitoring

DOI:  https://doi.org/10.3389/fneur.2025.1493201
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Mar 27. pii: S1570-0232(25)00128-X. [Epub ahead of print]1257 124576

A novel ultra-sensitive LC-MS/MS method for determination of elobixibat in human plasma; Application to a bioequivalence study on healthy volunteers.

Mamdouh R Rezk, Michael Soliman, Huda Shawky, Kamal A Badr, Mina Wadie.

  Towards a new era for alleviating chronic idiopathic constipation, elobixibat has been recently approved in Japan and completed phase III in clinical trials as a highly potent inhibitor for ileal bile acid transporter. This work provides the field of biomedical analysis and clinical studies with the first bioanalytical method for elobixibat quantitation in real human plasma. A new ultra-sensitive liquid chromatography-tandem mass spectrometric method was developed using elobixibat-d5 as an internal standard. First of all, several preliminary efforts were exerted and directed towards optimizing sample preparation procedures to extract the desired drug at a very low concentration level and to avoid any matrix interference or masking. The trials settled on adopting liquid-liquid extraction using methyl tertiary butyl ether after adding 200 μL of 10 % formic acid to 500 μL plasma sample. Chromatographic separation was then conducted using Kinetex® EVO C18 column with mobile phase composed of acetonitrile and 20 mM ammonium format acidified with 0.1 % formic acid in ratio of 80:20 (v/v) and pumped at 0.6 mL/min. Positive electrospray ionization was adopted for mass acquisition, operated in multiple reactions monitoring (MRM) mode at m/z 696 → 593.1 for elobixibat and m/z 701 → 598.1 for the IS. Full bioanalytical validation as per FDA guidance was done over a range of 20.0-1500.0 pg/mL. The proposed method was successfully exploited for determination of elobixibat in human plasma samples and extended to estimate the pharmacokinetic parameters after administration of a single oral dose of 5 mg elobixibat tablet to thirty healthy volunteers.

Keywords:  Bioequivalence study; Elobixibat; LC-MS/MS; Liquid-liquid extraction; Pharmacokinetic study

DOI:  https://doi.org/10.1016/j.jchromb.2025.124576
Anal Chem. 2025 Mar 31.

OzMALDI: A Gas-Phase, In-Source Ozonolysis Reaction for Efficient Double-Bond Assignment in Mass Spectrometry Imaging with Matrix-Assisted Laser Desorption/Ionization.

Josiah J Rensner, Hyojin Kim, Kiyoul Park, Edgar B Cahoon, Young Jin Lee.

Lipids make up an important class of biomolecules with diverse structures and varied chemical functions. This diversity is a major challenge in chemical analysis and limits our understanding of biological functions and regulation. A major way lipid isomers differ is by double-bond (db) position, and analyzing db-isomers is especially challenging for mass spectrometry imaging (MSI). Ozonolysis can be used to determine the db-position and has been paired with MSI before. However, previous techniques require increased analysis time to allow for gas-phase reactions within an ion trap or ion mobility cell or additional sample preparation time to allow for offline ozonation. Here, we introduce a new ozonolysis method inside the matrix-assisted laser desorption-ionization (MALDI) source, termed OzMALDI, that simultaneously produces ozonides from all unsaturated lipids. This allows us to determine db-positions without adding additional reaction time while maintaining the high mass resolution provided by Orbitrap MS. This new technique is especially effective at determining multiple db-positions in lipids containing polyunsaturated fatty acids, which is a limitation of many previous techniques. OzMALDI-MSI was applied to the analysis of rat brain and genetically engineered Camelina and soybean seed samples, demonstrating the utility of this method and uncovering novel biological information.

DOI: https://doi.org/10.1021/acs.analchem.5c00284
Rapid Commun Mass Spectrom. 2025 Jun 30. 39(12): e10023

Rapid Determination of 15 Herbicides in Blood and Soil by Direct Analysis in Real Time-Tandem Mass Spectrometry.

Zhou Yuan, Zhang Ying, Hou Xiaolong, Xue Chenyu, Zhang Wenfang, Qiao Jing, Liu Hua, Zhang Wenjin, Guo Yizhu.

   RATIONALE: The rampant abuse of herbicides in agricultural practices has resulted in frequent incidents of both unintended and deliberate poisoning, posing dual threats to ecosystems and human health. This underscores an urgent need for efficient herbicide detection methods.
METHODS: A direct analysis in real time coupled with tandem mass spectrometry (DART-MS/MS) method was developed for the simultaneous detection of 15 herbicides in blood and soil. Systematic optimization of mass spectrometric parameters established optimal detection conditions in multiple reaction monitoring (MRM) mode, with the ion source temperature set at 450°C. Following liquid-liquid extraction of blood or soil samples, the processed supernatant was analyzed directly.
RESULTS: The method demonstrated excellent linearity (R² ≥ 0.99) across a wide concentration range. Sensitivity was validated with limits of detection (LODs) of 1-20 ng/mL in blood and 1-10 ng/g in soil, and limits of quantification (LOQs) of 1-50 ng/mL in blood and 1-20 ng/g in soil. Satisfactory recovery rates and controlled matrix effects met toxicological requirements. In 2024, the method was successfully applied to four real cases of herbicide poisoning.
CONCLUSIONS: This study established the first DART-MS/MS method for simultaneous analysis of 15 herbicides in biological (blood) and environmental (soil) matrices. The complete analytical workflow required only 20 minutes from sample preparation to detection, significantly advancing forensic applications of DART-MS/MSwhile providing a reliable technical solution for herbicide screening in forensic investigations.

Keywords:  blood; direct analysis in real time‐tandem mass spectrometry (DART‐MS/MS); herbicide; soil

DOI:  https://doi.org/10.1002/rcm.10023
J Am Soc Mass Spectrom. 2025 Apr 03.

Norm ISWSVR Enhanced Data Repeatability and Accuracy in Large-Scale Targeted Quantification Metabolomics.

Jinpeng Bai, Chenxi Li, Mingmin Qian, Xian Ding, Qian Li, Yanhua Chen, Zhaoying Wang, Zeper Abliz.

  Targeted quantification metabolomics provides dynamic insights across various domains within the life sciences. Nevertheless, maintaining high-quality data obtained through liquid chromatography-mass spectrometry presents ongoing challenges. It is essential to develop normalization methods to correct for unwanted variations in metabolomic profiling such as batch effects and analytical drift. In this study, we assessed the normalization efficacy of Norm ISWSVR in targeted quantification metabolomics by comparing it with IS normalization and SERRF normalization. Consequently, Norm ISWSVR demonstrated exceptional efficacy in mitigating batch effects and reducing the relative standard deviation of quality control samples, in addition to correcting signal drift. Following normalization with Norm ISWSVR, the number of metabolites suitable for quantification increased with high precision. Collectively, Norm ISWSVR proves to be a robust and reliable method for enhancing data quality in targeted metabolomics, establishing itself as a promising approach for metabolomics research.

Keywords:  Norm ISWSVR; batch effects; signal drift; targeted metabolomics

DOI:  https://doi.org/10.1021/jasms.4c00467
Methods Mol Biol. 2025 ;2914 251-258

An LC-MS/MS Multiplexed Method to Quantify 14 Sphingolipids in Human Cerebrospinal Fluid (CSF).

Yadira Perez Paramo, Rathna Veeramachaneni, Emily Parkhurst, Dawn Dufield, W Rodney Mathews, Veronica G Anania.

  Sphingolipids are bioactive lipids comprised of a sphingoid base linked to various long-chain fatty acids and phosphocholine and, in some cases, may also contain one or more sugars. A few species of sphingolipids share similar structures and may even have identical chemical composition but differ in their biological function. For this reason, they can only be accurately quantified using liquid chromatography coupled with mass spectrometry (LC-MS). This technique can separate sphingolipids based on their hydrophobicity and quantify them based on their mass-to-charge ratio (m/z). This is especially important when measuring isomeric species that have identical m/z but differ in their retention time. Here, we describe the development of a new LC-MS method to measure 14 sphingolipid species, including 7 ceramides (C14, C16, C18, C20, C24, C18:1, C24:1), 4 glycosphingolipids (C16 Glucosyl(ß) Ceramide (d18:1/16:0, C16 Galactosyl(ß) Ceramide (d18:1/16:0), C16 Lac Cer, C18 Lac Cer) and 3 sphingosines (Sphingosine (Sph), Glucosyl(ß) Sphingosine (d18:1), Galactosyl(ß) Sphingosine (d18:1)) in human cerebrospinal fluid (CSF). Using this 7-min method, chromatographic resolution of isomeric species glucosylceramide (Glc Cer) and galactosylceramide (Gal Cer) was achieved. This resolution is important as these two sphingolipid classes have distinct biological functions with important implications in neurological diseases like multiple sclerosis.

Keywords:  CSF; Gal Cer; Glc Cer; Isomers; LC-MS; Neurodegenerative diseases; Sphingolipids

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_18
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Mar 28. pii: S1570-0232(25)00130-8. [Epub ahead of print]1257 124578

Pentafluorobenzyl bromide - A versatile derivatization agent in chromatography and mass spectrometry: II. Analysis of organic acids and bases, and comparison with other perfluorinated reagents.

Dimitrios Tsikas.

  Analytical derivatization is an important for the vast majority of substances an indispensable sample preparation step for their quantitative GC-MS and GC-MS/MS analysis in biological samples. Pentafluorobenzyl bromide (PFB-Br), pentafluorobenzoyl chloride (PFB-COCl), pentafluorobenzyl hydroxylamine (PFB-NHNH2), pentafluorophenyl hydrazine (PFPh-ONH2), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric anhydride (HFBA) are versatile derivatization reagents in analytical chemistry. In the present work, the utility of the above mentioned derivatization reagents for the GC-MS analysis of carboxylic, aldehydic, hydroxylic and amine groups containing analytes including amino acids is reviewed and discussed. Derivatization requires different conditions for solvents, reaction temperature and time, and possibly for catalysts. The perfluorinated derivatives are electrically neutral and best soluble in water-immiscible organic solvents such as toluene. Under negative-ion chemical ionization (NICI) conditions, the perfluorinated derivatives readily and abundantly ionize that allows for sensitive analysis. In addition, the perfluorinated analyte derivatives emerge earlier from GC columns than protiated, thus enabling shorter analysis times. Externally added 2H-, 13C-, 15N and 18O-isotopologs for use as internal standards undergo similar changes during derivatization, extraction by organic solvents, ionization in the ion-source of GC-MS apparatus and have almost identical retention times with the analytes. Due to selective analytical derivatization, almost all classes of endogenous and exogenous low-molecular-mass analytes, including drugs and inorganic anions such as nitrite, nitrate, carbonate, and (pseudo)halogenides, become accessible to quantitative GC-MS and GC-MS/MS analysis. Thanks the high sensitivity of quantitative analytical methods based on GC-MS and GC-MS/MS, very low amounts of perfluorinated derivatization reagents are consumed. In consideration of the enormously high global warming potential (GWP) of F-containing derivatization reagents, this article discussed a potential abandonment of the use of perfluorinated reagents and their replacement by F-free reagents in GC-MS and GC-MS/MS.

Keywords:  Derivatization; Fluorine; GC–MS; GC–MS/MS; Global warming potential; Perfluorinated reagents

DOI:  https://doi.org/10.1016/j.jchromb.2025.124578
Methods Mol Biol. 2025 ;2914 229-250

Elucidation of the Human Cerebrospinal Fluid Metabolome by Ion Mobility Mass Spectrometry and NMR Spectroscopy.

Jannik Sprengel, Georgy Berezhnoy, Christoph Trautwein.

  In the last decades, the prevalence of neurodegenerative disorders such as Parkinson's disease, dementia and stroke has significantly increased. The lack of early-stage biomarkers hereby is a major reason for poor outcomes and limited treatment options. Metabolism alterations are a major driver and readout of neurological implications; however, it can be assessed only to a very minor extent by non-invasive imaging technologies. By contrast, typical diagnostic biofluids like serum or urine reflect metabolic changes on the systemic scale and are not specific to the brain. That's why the analysis of cerebrospinal fluid (CSF) using metabolomic techniques offers a great perspective to better stratify patients with neurological implications. However, as metabolites can readily degrade and CSF collection in a clinical environment is challenging, several confounders have to be considered. Furthermore, no single technique can measure the full CSF metabolome. Hence in this work we used directly collected and quenched CSF for a multi-modal metabolomics profiling approach to define the ex vivo baseline of the human CSF metabolome by robust Nuclear Magnetic Resonance (NMR) spectroscopy and latest state of the art liquid chromatography (LC) ion mobility spectrometry mass spectrometry (IMS-MS).

Keywords:  CSF; Liquor; Mass spectrometry; Metabolomics; NMR spectroscopy; timsTOF

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_17
Clin Chim Acta. 2025 Mar 26. pii: S0009-8981(25)00141-X. [Epub ahead of print]572 120262

Establishment of the isotope dilution-gas chromatography-tandem mass spectrometry (ID-GC/MS) method for accurate quantification of phenylalanine and its application to value assignment of external quality assessment samples.

Zhizheng Li, Zhonggan Jin, Ming Zong.

   OBJECTIVES: Phenylalanine (Phe) was an essential amino acid that was crucial for diagnosing phenylketonuria (PKU) and managing their blood Phe levels. Currently, there was no JCTLM-recognized reference method for measuring Phe internationally. To standardize the measurement of serum Phe, our primary goal was to establish an accurate quantitative method based on isotope dilution combined with gas chromatography-mass spectrometry (ID-GC/MS) for the quantitative detection of Phe in human serum, and its performance was thoroughly validated.
METHODS: Serum samples were pre-treated using acetonitrile protein precipitation. After derivatization with N-(Methyl)-N-(trimethylsilyl)trifluoroacetamide (MSTFA), serum Phe content was quantitatively detected using the ID-GC/MS method. Mass spectrometry analysis was conducted using the SIM monitoring mode. The analytical performance and application of the new method were investigated. External quality assessment (EQA) samples were tested using the established method, and their accuracy was compared with clinical laboratory results.
RESULTS: The total detection time for Phe using the established method was 7 min. This method was capable of quantifying Phe within the range of 7.39 to 1188.81 μmol/L, with a limit of quantitation of 3.03 μmol/L. The intra-assay and inter-assay coefficients of variation (CV) were both ≤2.5 %, and the spike recovery rate ranged from 97.64 % to 100.72 %, and a relative expanded uncertainty was ≤3.0 % (k = 2). The method was free from interference, matrix effect and carry-over. Moreover, the ID-GC/MS measurement procedure was successfully applied to measure Phe in serum samples and to assign value of EQA samples.
CONCLUSIONS: This study successfully established an accurate quantitative method for detecting serum Phe concentrations using ID-GC/MS technology and it could support EQA management for Phe testing, providing traceable target values for samples.

Keywords:  Accurate quantitative method; Amino acids; Isotope dilution-gas chromatography-tandem mass spectrometry; Laboratory medicine; Phenylalanine

DOI:  https://doi.org/10.1016/j.cca.2025.120262
Anal Chem. 2025 Apr 01.

Rapid MALDI-MS/MS-Based Profiling of Lipid A Species from Gram-Negative Bacteria Utilizing Trapped Ion Mobility Spectrometry and mzmine.

Edward Rudt, Matti Froning, Steffen Heuckeroth, Lucas Ortmann, Julia Diemand, Linus Hörnschemeyer, Alexander Pleger, Max Vinzelberg, Robin Schmid, Tomáš Pluskal, Ulrich Dobrindt, Heiko Hayen, Ansgar Korf.

Lipid A, a crucial component of lipopolysaccharides (LPS), plays a pivotal role in the pathogenesis of Gram-negative bacteria. Lipid A patterns are recognized by mammals and can induce immunostimulatory effects. However, the outcome of the interaction is highly dependent on the chemical composition of individual lipid A species. The diversity of potential fatty acyl and polar headgroup combinations in this complex saccharolipid presents a significant analytical challenge. Current mass spectrometry (MS)-based lipid A methods are focused on either direct matrix-assisted laser desorption/ionization (MALDI)-MS screening or comprehensive structural elucidation by tandem mass spectrometry (MS/MS) hyphenated with separation techniques. In this study, we developed an alternative workflow for rapid lipid A profiling covering the entire analysis pipeline from sample preparation to data analysis. This workflow is based on microextraction and subsequent MALDI-MS/MS analysis of uropathogenic Escherichia coli utilizing trapped ion mobility spectrometry (TIMS), followed by mzmine data processing. The additional TIMS dimension served for enhanced sensitivity, selectivity, and structural elucidation through mobility-resolved fragmentation via parallel accumulation-serial fragmentation (PASEF) in parallel reaction monitoring (prm)-mode. Furthermore, mzmine enabled automated MS/MS acquisition by adapting the spatial ion mobility-scheduled exhaustive fragmentation (SIMSEF) strategy for MALDI spot analysis. It also facilitated robust lipid A annotation through a newly developed extension of the rule-based lipid annotation module, allowing for the custom generation of lipid classes, including specific fragmentation rules. In this study, the first publication of lipid A species' collision cross section (CCS) values is reported, which will enhance high-confidence lipid A annotation in future studies.

DOI: https://doi.org/10.1021/acs.analchem.4c05989
Biomed Chromatogr. 2025 May;39(5): e70065

Optimizing Mirabegron Stability in Human Plasma: Method Development, Validation Through Integration of Esterase Inhibitors and Stabilizers.

Santosh Tawari, Ujashkumar Shah.

  The stability of mirabegron in human plasma is addressed in this study. The susceptibility of Mirabegron to enzymatic and oxidative degradation requires the integration of esterase inhibitors and stabilizers into the analytical process. Esterase inhibitors, including sodium fluoride and dichlorvos, as well as stabilizing agents such as malic acid, ascorbic acid, acetic acid, citric acid and sodium bicarbonate were also investigated to mitigate this issue. Samples were prepared through solid-phase extraction (SPE) utilizing Strata-X cartridges. The optimization of chromatographic separation was conducted using a C18 Kromasil column with a solvent mixture consisting of ammonium formate and acetonitrile. Detection employed electrospray ionization (ESI) in positive ionization mode, focusing on MRM transitions of 397.2 → 260.1 for mirabegron and 402.2 → 260.1 for mirabegron D5. The method exhibited an accuracy range of 95.67% to 102.85% and precision of 0.52% to 2.31% for a linearity ranged from 0.100 to 102.496 ng/mL. An advantage of this method is its stability, requiring only a minimal plasma volume of 100 μL, a quick runtime of 3 min, and a high recovery rate of 92.93%, making it highly efficient and reliable for bioequivalence testing, therapeutic monitoring and pharmacokinetic studies.

Keywords:  LCMS; human plasma; mirabegron; mirabegron D5; sodium fluoride

DOI:  https://doi.org/10.1002/bmc.70065
Methods Mol Biol. 2025 ;2914 203-212

Mass Spectrometry Applied to Human Cerebrospinal Fluid Lipidome (II).

Laura Millán, Joaquín Fernández-Irigoyen, Enrique Santamaría, Rebeca Mayo.

  Lipidomics aims at characterizing lipid profiles and their biological role with respect to protein expression involved in lipid metabolism. Specifically, cerebrospinal fluid (CSF) lipidomics is offering a new perspective in the search for surrogate biomarkers to facilitate early diagnosis of psychiatric and neurodegenerative diseases. In this chapter, we describe a nontargeted approach to profile lipid molecular species present in human CSF using ultrahigh-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (UPLC-ESI-ToF-MS). This workflow complements the toolbox useful for the exploration and monitoring of neurodegenerative mechanisms associated with dysregulation in lipid metabolism.

Keywords:  CSF; Lipidomics; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_15
Anal Chem. 2025 Apr 04.

Conversion Reaction of Stable-Isotope Oxygen Labeling of Carboxylic Acids for Accurate Screening LC-MS/MS Assay: Application of Behavioral Changes of Short-Chain Fatty Acids in Sports Athletes under Exercise Loading.

Aoi Ichikawa, Takahiro Takayama, Chihiro Kojima, Shumpei Fujie, Motoyuki Iemitsu, Koichi Inoue.

Short-chain fatty acids (SCFAs) have attracted considerable interest as potential biomarkers, therapeutic targets, and nutritional factors in athletic training. SCFAs are typically produced by the intestinal microbiome and exhibit various structural forms, including linear- and branched-chain types. In particular, branched-chain SCFAs have been associated with muscle metabolism during exercise loading. Consequently, accurate and efficient analytical methods are essential for identifying these biomarkers. Liquid chromatography-tandem mass spectrometry is a suitable and accurate technique for SCFA analysis; however, stable isotope calibrations are required for all analytes. Because of technological limitations, the available species are restricted to certain types of SCFAs. To address this issue, this study performed a simple conversion reaction involving the incorporation of 18O into the carboxyl group. Specifically, oxygen atoms in the carboxyl groups were substituted with 18O sourced from commercially available H218O. An SCFA mixture standard solution was successfully labeled under optimized conditions, and the SIL purity and amount were sufficient for isotope dilution (95.2-96.9%, 250 assays using 10 μL of H218O). Moreover, no reversion to 16O was observed during storage or analysis. Analytical validation was performed in human serum using the substituted isotopic standard mixture, achieving good accuracy (90-110%) and precision (<10% relative standard deviation) across three concentration levels. Finally, changes in SCFA patterns were examined in athletes during exercise loading.

DOI: https://doi.org/10.1021/acs.analchem.4c05872
Nat Methods. 2025 Mar 28.

Denoising Search doubles the number of metabolite and exposome annotations in human plasma using an Orbitrap Astral mass spectrometer.

Fanzhou Kong, Tong Shen, Yuanyue Li, Amer Bashar, Susan S Bird, Oliver Fiehn.

Chemical exposures may affect human metabolism and contribute to the etiology of neurodegenerative disorders such as Alzheimer's disease. Identifying these small metabolites involves matching experimental spectra to reference spectra in databases. However, environmental chemicals or physiologically active metabolites are usually present at low concentrations in human specimens. The presence of noise ions can substantially degrade spectral quality, leading to false negatives and reduced identification rates. In response to this challenge, the Spectral Denoising algorithm removes both chemical and electronic noise. Spectral Denoising outperformed alternative methods in benchmarking studies on 240 tested metabolites. It improved high confident compound identifications at an average 35-fold lower concentrations than previously achievable. Spectral Denoising proved highly robust against varying levels of both chemical and electronic noise even with a greater than 150-fold higher intensity of noise ions than true fragment ions. For human plasma samples from patients with Alzheimer's disease that were analyzed on the Orbitrap Astral mass spectrometer, Denoising Search detected 2.5-fold more annotated compounds compared to the Exploris 240 Orbitrap instrument, including drug metabolites, household and industrial chemicals, and pesticides.

DOI: https://doi.org/10.1038/s41592-025-02646-x
Anal Chim Acta. 2025 May 08. pii: S0003-2670(25)00219-3. [Epub ahead of print]1350 343825

Qualitative and quantitative lipidomic profiling of cardiolipin, oxidized cardiolipin, and monolysocardiolipin from human skeletal muscle by UPLC-Orbitrap-MSn.

Zhi-Xin Yuan, Josephine M Egan, Arsun Bektas, Qu Tian, Alexey Lyashkov, Luigi Ferrucci, Christopher E Ramsden, Ruin Moaddel.

   BACKGROUND: Cardiolipins (CL) are a mitochondria-specific family of phospholipids that play central roles in mitochondrial function. Imbalance in CL metabolism, especially excessive CL oxidation, leads to mitochondrial dysfunction, apoptosis, and inflammation, contributing to age-related diseases. As of yet no comprehensive methods have been developed to assess CL, oxidized CL (oxCL), and monolyso-CL (MLCL) species.
RESULTS: To fill this critical research gap, we combined untargeted and targeted lipidomic approaches to analyze CL species in human skeletal muscle samples. The method enabled in-depth structural characterization using exact mass measurement followed by multistage fragmentation (MSn) to achieve unequivocal structural elucidation at the molecular species level and sn-position level for some species. This novel methodology identified intact mono- and di-oxygenated L4CL species and allowed the differentiation of isomeric 9/13-HODE-L3CL and 9(10)/12(13)-EpOME-L3CL with unprocessed total lipid extracts. Overall, 220 molecular species (125 CL, 30 oxCL, and 65 MLCL) were detected. Our method includes a quantitation strategy that leveraged on establishing three-leveled matrix-matched calibration curves normalized by using internal standard M4CL and its isotopologues M+1 (13C-M4CL) and M+3 (13C3-M4CL) respectively. The analytical performance was also evaluated and found to be highly sensitive with LLOQs at fmol levels and reproducible with precision RSD <20 % for the majority.
SIGNIFICANCE AND NOVELTY: This is the first reported method to simultaneously provide broad coverage of CL, oxCL, and MLCL species from a single injection of total lipid extract in a complex biological sample. This method is also the first to demonstrate the presence of sn-positional isomers of CL in human skeletal muscle. It has been successfully applied to a pilot study of skeletal muscle biopsies and provided meaningful results. We anticipate the methodology will facilitate investigations of the cardiolipin lipidome leading to a better understanding of the complex and highly interactive biological processes that regulate mitochondria function and expand minor cardiolipin targets including oxCL and MLCL for biomarker discovery.

Keywords:  Cardiolipins; Human skeletal muscle; Lipidomics; Monolysocardiolipins; Oxygenated cardiolipins; RP-UPLC/HR-MS(n)

DOI:  https://doi.org/10.1016/j.aca.2025.343825
J Anal Toxicol. 2025 Apr 03. pii: bkaf027. [Epub ahead of print]

Synthetic cannabinoids receptor agonists in oral fluid: development of a dispersive liquid-liquid microextraction (DLLME) method with liquid chromatography-mass spectrometry detection.

Henrique Silva Bombana, Gabriela de Paula Meirelles, Rodrigo Alves de Oliveira, Vilma Leyton, Mauricio Yonamine.

  Synthetic cannabinoid receptor agonists (SCRAs) comprise a class of new psychoactive substances (NPS) that rank second in terms of notified substances to the United Nations Office on Drugs and Crime. Moreover, SCRAs are the most prevalent NPS in Brazilian territory. Given the risks they pose to public health, there is a pressing need to develop simple and rapid sample preparation methods in alternative biological matrices that are easy to handle and collect, such as oral fluid (OF). In this study, dispersive liquid-liquid microextraction (DLLME) was employed to determine twelve SCRAs in OF. For 200 µL of sample (mixture of OF and Quantisal™ buffer), 200 µL of ice-cold acetonitrile were used as the dispersive solvent, and 100 µL of ethyl acetate were used as the extraction solvent. The limits of detection ranged from 0.5 to 2 ng/mL, while the limits of quantification were 2 ng/mL for ADB-FUBIATA and 1 ng/mL for the other analytes. The working range was 1-100 ng/mL, except for ADB-FUBIATA, which had a range of 2-100 ng/mL. The coefficients of variation for quantified analytes were <11.3% for within-run precision, <12.6% for between-run precision, and <15.8% for accuracy across all controls. The developed method was applied to six suspected samples, and one sample yielded a positive result with 39.9 ng/mL of MDMB-4en-PINACA, the most prevalent SCRA in São Paulo State, Brazil.

Keywords:  DLLME; LC-MS/MS; oral fluid; synthetic cannabinoid receptor agonists

DOI:  https://doi.org/10.1093/jat/bkaf027
Anal Chem. 2025 Apr 04.

Data-Independent Acquisition Coupled with Electron-Activated Dissociation for In-Depth Structure Elucidation of the Fatty Acid Ester of Hydroxy Fatty Acids.

Yuto Kurizaki, Yuki Matsuzawa, Mikiko Takahashi, Hiroaki Takeda, Mayu Hasegawa, Makoto Arita, Junki Miyamoto, Hiroshi Tsugawa.

Fatty acid esters of hydroxy fatty acid (FAHFAs) are a biologically important class of lipids known for their anti-inflammatory and antidiabetic effects in animals. The physiological activity of FAHFAs varies depending on the length of the carbon chain, number and position of double bonds (DBs), and position of the hydroxyl (OH) group. Moreover, gut bacteria produce FAHFAs with more diverse structures than those produced by the host, which necessitates a FAHFA-lipidomics approach grasping their diverse structures to fully understand the physiological and metabolic significance of FAHFAs. In this study, we developed a methodology for the in-depth structural elucidation of FAHFAs. First, FAHFAs were enriched by using a solid-phase extraction (SPE) system coated with titanium and zirconium dioxide, which separated these analytes from neutral lipids and phospholipids. The fractionated metabolites were then derivatized using N,N-dimethylethylenediamine (DMED) to facilitate FAHFA detection in the positive ion mode of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A data-independent acquisition technique known as sequential window acquisition of all theoretical mass spectra (SWATH-DIA) was used to collect sequential MS/MS spectra of the DMED-derivatized fatty acid metabolites. Structural elucidation was based on fragment ions generated by electron-activated dissociation (EAD). DMED-FAHFAs were annotated using the newly updated MS-DIAL program, and FAHFA isomers were quantified using the MRMPROBS program, which quantifies lipids based on SWATH-MS/MS chromatograms. This procedure was applied to profile the FAHFAs present in mouse fecal samples, characterizing seven structures at the molecular species level, 63 structures at the OH-position-resolved level, and 15 structures at both the DB- and OH-position-resolved levels, using the MS-DIAL program. In the MRMPROBS analysis, 2OH and 3OH hydroxy fatty acids with more than 20 carbon atoms were predominantly expressed, while 5OH-13OH hydroxy fatty acids with 16 or 18 carbon atoms were the major components, abundant at positions 5, 7, 9, and 10. Furthermore, age-related changes in FAHFA isomers were also observed, where FAHFA 4:0/2O(FA 26:0) and FAHFA 16:0/10O(FA 16:0) significantly increased with age. In conclusion, our study offers a novel LC-SWATH-EAD-MS/MS technique with the update of computational MS to facilitate in-depth structural lipidomics of FAHFAs.

DOI: https://doi.org/10.1021/acs.analchem.4c06736
J Proteome Res. 2025 Apr 04.

metLinkR: Facilitating Metaanalysis of Human Metabolomics Data through Automated Linking of Metabolite Identifiers.

Andrew Patt, Iris Pang, Fred Lee, Chiraag Gohel, Eoin Fahy, Vicki Stevens, David Ruggieri, Steven C Moore, Ewy A Mathé.

  Metabolites are referenced in spectral, structural and pathway databases with a diverse array of schemas, including various internal database identifiers and large tables of common name synonyms. Cross-linking metabolite identifiers is a required step for meta-analysis of metabolomic results across studies but made difficult due to the lack of a consensus identifier system. We have implemented metLinkR, an R package that leverages RefMet and RaMP-DB to automate and simplify cross-linking metabolite identifiers across studies and generating common names. MetLinkR accepts as input metabolite common names and identifiers from five different databases (HMDB, KEGG, ChEBI, LIPIDMAPS and PubChem) to exhaustively search for possible overlap in supplied metabolites from input data sets. In an example of 13 metabolomic data sets totaling 10,400 metabolites, metLinkR identified and provided common names for 1377 metabolites in common between at least 2 data sets in less than 18 min and produced standardized names for 74.4% of the input metabolites. In another example comprising five data sets with 3512 metabolites, metLinkR identified 715 metabolites in common between at least two data sets in under 12 min and produced standardized names for 82.3% of the input metabolites. Outputs of MetLInR include output tables and metrics allowing users to readily double check the mappings and to get an overview of chemical classes represented. Overall, MetLinkR provides a streamlined solution for a common task in metabolomic epidemiology and other fields that meta-analyze metabolomic data. The R package, vignette and source code are freely downloadable at https://github.com/ncats/metLinkR.

Keywords:  databases; lipidomics; metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.4c01051
J Am Soc Mass Spectrom. 2025 Apr 03.

Evaluating Ion Mobility Data Acquisition, Calibration, and Processing for Small Molecules: A Cross-Platform Assessment of Drift Tube and Traveling Wave Methodologies.

James N Dodds, Lucie C Ford, Jack P Ryan, Amie M Solosky, Ivan Rusyn, Erin S Baker.

As ion mobility spectrometry (IMS) separations continue to be added to analytical workflows due to their power in environmental and biological sample analyses, harmonization and capability understanding between existing and newly released instruments are desperately needed. Developments in IMS platforms often exhibit focus on increasing resolving power (Rp) to better separate molecules of similar structure. While the additional separation capacity is advantageous, ensuring these developments coincide with appropriate data extraction and analysis methods is imperative to ensure routine adoption. Herein, we assess the performance of the MOBILion MOBIE in relation to a commercially available drift tube IMS-MS, the Agilent 6560, and evaluate feature extraction and analysis pipelines. Both instruments were operated using matched conditions when possible, and performance metrics of scan speed, Rp, limits of detection (LOD), and propensity for isomer separation via LC-IMS-MS were evaluated. Similar scan speeds pertaining to IMS-MS frame generation were noted for both platforms, and collision cross section (CCS) values for the MOBIE were generally within ≤ 1% difference from previously reported drift tube values. Both platforms were also able to generate quantitative data (comparable limits of detection) in experiments with perfluoroalkyl substances (PFAS) mixtures in a cell-based model (both medium and cell lysates), as demonstrated in Skyline with adjusted mobility filtering parameters. Higher Rp was, however, noted on the MOBIE in comparison to the 6560 (200-300 vs 45-60 CCS/ΔCCS without data processing), allowing the detection of more PFAS isomers and indicating promise toward future applications in chemical exposomics studies and biomarker discovery when molecules exhibit similar structures.

DOI: https://doi.org/10.1021/jasms.5c00056
J AOAC Int. 2025 Apr 03. pii: qsaf031. [Epub ahead of print]

Development and Validation of a Sensitive LC-MS/MS Method for Simultaneous Determination of Several Nitrosamines in Large Volume Parenteral.

Lukas Jost, Shiwei Zhou, Frank Sprau, Payden Trujillo, Annie Wan, Lucas Shaner, Bilig Sechin, Lars Gläser, David Young, Percy Kampeis, Weichun Yang.

   BACKGROUND: Nitrosamines have gained significant attention in pharmaceutical industry. However, due to the significant daily dosage of large volume parenteral (LVP), the detection limit for these products should be in the ng per liter range, which many published methods cannot achieve.
OBJECTIVE: This study aimed to develop and validate a sensitive LC-MS/MS method for the simultaneous determination of several nitrosamines in LVP drug products.
METHOD: Nitrosamines and related internal standards were separated on a Waters ACQUITY UPLC HSS T3 (100 × 2.1 mm, 1.8 µm) column on a LC-MS/MS system with gradient elution. Prior to the LC injection, a Carbon A solid phase extraction (SPE) column was utilized to pretreat the test solutions. The mobile phase was composed of 0.1% formic acid in water as mobile phase A, and neat methanol as mobile phase B. Analytes were detected via multiple reaction monitoring (MRM) mode and quantitated against internal reference standards with a quantitation limit of 2.5 ng/L for N-nitrosodimethylamine (NDMA), 0.75 ng/L for other Nitrosamine analytes.
RESULTS: The LC-MS/MS method was able to separate all analytes of interest by gradient elution within 15 min. The method was validated according to the guidelines described in the International Conference on Harmonization guideline ICH Q2(R2). The LOQ for NMDA is 2.5 ng/L, while for other nitrosamines, it is 0.75 ng/L in peritoneal dialysis and saline matrices. For haemofiltration solution, the LOQ is 1.0 ng/L for NDMA and 0.3 ng/L for other nitrosamines. The RSD% (n = 9) of the recovery did not exceed 25% in the method accuracy evaluation. A comparative testing in three labs revealed that all three laboratories are capable of accurately measuring nitrosamine levels at 10 ng/L within the intricate LVP matrix.
CONCLUSIONS: The LC-MS/MS method for several nitrosamines was successfully developed, validated, and demonstrated to be accurate, robust, and specific.
HIGHLIGHTS: The performance of this method can reach single digit ng/L level in LVP matrices. Comparative testing was conducted in three laboratories located in China, Germany, and the United States to ensure reproducibility of the method.

Keywords:  LC-MS/MS; Nitrosamine impurities; full method validation; large volume parenteral (LVP); trace level analysis

DOI:  https://doi.org/10.1093/jaoacint/qsaf031
J Agric Food Chem. 2025 Mar 30.

3D Mass Spectrometry Imaging as a Novel Screening Method for Evaluating Biocontrol Agents.

Justyna Szulc, Tomasz Grzyb, Beata Gutarowska, Joanna Nizioł, Sumi Krupa, Tomasz Ruman.

  The aim of this study was to evaluate innovative mass spectrometry imaging (MSI) for determining the metabolic potential of selected soil bacteria from the genera Bacillus and Priestia in the presence of the phytopathogen Fusarium. This research marks the first application of direct 3D MSI that to visualize interactions between potential biocontrol agents and plant pathogens on agar medium. The LARAPPI/CI-3D-MSI (Laser-Assisted Remote Atmospheric Pressure Imaging/Chemical Ionization-3D Mass Spectrometry Imaging) setup provided valuable insights into the compounds produced by the tested microorganisms, revealing their spatial distributions and their ability to diffuse into the agar medium. Subsequently, a Pathway Impact Analysis of Metabolites was conducted. Ion images based on ultrahigh resolution mass spectrometry data were obtained, including for potentially bioactive compounds. Statistical analysis of the entire MS data set showed that the metabolites identified for Bacillus licheniformis samples were distinctly separated from the Priestia megaterium samples and could be helpful tools for screening biocontrol strains. The LARAPPI/CI MSI technique offers significant advantages over existing MSI methods. Further research using this technology could help validate the effectiveness of various biopreparations and contribute to enhancing the quality of biological plant protection products available on the market.

Keywords:  2D/3D biological sample imaging; antagonism; biocontrol agents; mass spectrometry imaging; metabolomic analysis; phytopathogens

DOI:  https://doi.org/10.1021/acs.jafc.5c00349