bims-metlip 2025-08-17 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–08–17
twenty-two papers selected by
Sofia Costa, Matterworks

Simple and sensitive LC-MS analysis of short chain fatty acids in cecum content and feces in mice: A derivatization-free approach.
Reverse Spectral Search Reimagined: A Simple but Overlooked Solution for Chimeric Spectral Annotation.
Multi-Modal Spatial Metabolomics with Mid-Infrared Microscope Guided Mass Spectrometry Imaging.
Computationally unmasking each fatty acyl C=C position in complex lipids by routine LC-MS/MS lipidomics.
Development and Validation of a LC-MS/MS Method for Ripretinib and Its Metabolite: Example of a Journey From Laboratory Bench to Routine Application With a Greenness Assessment.
Validation and clinical application of an LC-MS/MS method designed to simultaneously measure seven second-line TB drugs and two metabolites in human lung tissue.
Evaluating Novel Direct Injection Liquid Chromatography-Mass Spectrometry Method and Extraction-Based Workflows for Untargeted Lipidomics of Extracellular Vesicles.
Development of a simultaneous analysis method for 13 neonicotinoids and related insecticides in whole blood by LC-MS/MS using a Novum simplified liquid extraction column.
Enhancing Lipidomics With High-Resolution Ion Mobility-Mass Spectrometry.
Simple and robust analysis of cefuroxime in human plasma and bone tissues by LC-MS/MS.
Development and validation of a 'dilute and shoot' LC-MS/MS method in urine suitable for screening and confirmation analyses in a clinical setting.
Development and validation of simultaneous quantification of total and free clozapine and its two metabolites in human plasma using ultra high-performance liquid chromatography coupled to tandem mass spectrometry.
Measurement of Total and Free Cefiderocol Concentrations in Human Plasma by UHPLC-MS/MS: Application to Patients With Difficult-To-Treat Gram-Negative Bacterial Infections.
Analysis of per-and polyfluorinated alkyl substances by chromatography-mass spectrometry: A review.
Development of a Liquid Chromatography-Tandem Mass Spectrometry Method for Oxylipin Analysis and Its Application to Children's Plasma.
Ultra-high throughput mass spectrometry with ultra high-speed separations: differential mobility spectrometry-acoustic ejection mass spectrometry (DAEMS).
An automated software-assisted approach for exploring metabolic susceptibility and degradation products in macromolecules using high-resolution mass spectrometry.
MetaboliticsDB: A Database of Metabolomics Analyses.
Development of a sensitive UPLC-MS/MS method for simultaneous determination of 15 Bisphenol A analogues in human serum and urine: Application of paired samples and global exposure assessment.
The Role of LC-MS in Profiling Bioactive Compounds from Plant Waste for Cosmetic Applications: A General Overview.
Quantitation of amylase/trypsin-inhibitors in barley using targeted LC-MS/MS.
Long-Term System Suitability Evaluation for Mass Accuracy in the Analysis of Small Molecules by High-Resolution Mass Spectrometry.

J Chromatogr A. 2025 Aug 09. pii: S0021-9673(25)00608-9. [Epub ahead of print]1760 466263

Simple and sensitive LC-MS analysis of short chain fatty acids in cecum content and feces in mice: A derivatization-free approach.

Soo Takasu, Yumi Nagasaki, Juri Koizumi, Tetsuo Koshizuka, Yukihiro Esaka.

  Short-chain fatty acids (SCFAs), such as acetic, propionic, and butyric acid, are important biomarkers that reflect gut microbiota composition, disease progression, and overall health. Conventional SCFA analysis typically involves derivatization prior to liquid or gas chromatography. However, derivatization is challenging due to the volatility of SCFAs and interference from similar carboxylic acids in biological samples and often requires complex purification steps. Consequently, the development of derivatization-free liquid chromatography-mass spectrometry (LC-MS) methods is desirable, although direct LC-MS analysis of intact SCFAs often suffers from limited sensitivity and accuracy. In this study, we have developed a derivatization-free LC-MS method for the analysis of major SCFAs (C2-C4) in the cecum content and feces of mice. Separation was performed using a mixed-mode column combining hydrophilic interaction chromatography and anion-exchange chromatography. We found that ammonium formate, required for separation, significantly suppressed SCFA signal intensity, whereas its replacement by ammonium fluoride prevented this suppression. Furthermore, we have demonstrated that the background noise in biological sample analysis can be reduced by selective reaction monitoring (SRM) mode, in which the same m/z value was set for both the precursor and product ions, as compared with selective ion monitoring (SIM) mode. The method developed in this study showed good validation values and sensitivity in the quantification of targeted SCFAs in mouse biological samples, demonstrating the excellent practicability of this method and making it a useful tool for research on the gut microbiome.

Keywords:  Ammonium fluoride; Gut microbiota; Hydrophilic interaction chromatography (HILIC); LC-MS analysis; Short-chain fatty acids (SCFAs)

DOI:  https://doi.org/10.1016/j.chroma.2025.466263
Anal Chem. 2025 Aug 15.

Reverse Spectral Search Reimagined: A Simple but Overlooked Solution for Chimeric Spectral Annotation.

Shipei Xing, Vincent Charron-Lamoureux, Haoqi Nina Zhao, Yasin El Abiead, Mingxun Wang, Pieter C Dorrestein.

The exponential growth of untargeted metabolomics data, now reaching billions of mass spectra in public repositories, benefits from reannotation strategies for data reuse. While tandem mass spectrometry (MS/MS) library matching remains the gold standard, annotation workflows face a critical bottleneck: chimeric spectra. These composite spectra, arising from simultaneous fragmentation of multiple precursor ions, compromise the ability to annotate MS/MS spectra against nonchimeric reference spectra. Here, we demonstrate that an enhanced version of reverse spectral search, a principle first introduced in 1975 but largely overlooked, provides a simple, computationally efficient solution, rescuing up to 62% more metabolite annotations in benchmark data sets while maintaining stringent quality control. The enhanced reverse spectral search is now added to the GNPS ecosystem. Source codes can be accessed at https://github.com/Philipbear/reverse_search.

DOI: https://doi.org/10.1021/acs.analchem.5c02047
J Vis Exp. 2025 Jul 25.

Multi-Modal Spatial Metabolomics with Mid-Infrared Microscope Guided Mass Spectrometry Imaging.

Minqian Wei, Haining Wang, Yirong Zeng, Dan Ye, Gangqi Wang, Lixue Shi.

Spatial metabolomics is a rapidly evolving field to map the distribution of metabolites within tissues, organs, and even single cells. This approach provides contextual metabolic information, which is critical for understanding the biochemical heterogeneity of biological systems. Mid-infrared (MIR) imaging and mass spectrometry imaging (MSI) have emerged as powerful approaches for spatial metabolomics, each offering unique and complementary advantages. In this study, we present a workflow for performing MIR imaging and matrix-assisted laser desorption ionization MSI (MALDI-MSI) on the same tissue section, encompassing experimental procedures, imaging co-registration, data integration, and bioinformatics analysis. MIR imaging is employed as the upstream modality, allowing for non-destructive biochemical analysis of tissues. Subsequently, a matrix is deposited in the tissue for MALDI-MSI, guided by the spatial information obtained from MIR imaging. Images are integrated following co-registration, enabling multi-modal spatial metabolomics analysis using advanced bioinformatics tools such as Seurat. The integration of MIR imaging and MSI represents a transformative advancement in spatial metabolomics, offering unprecedented opportunities to explore the spatial and chemical complexity of metabolic processes in both health and disease. This multi-modal approach holds significant potential for driving innovations in biomarker discovery, disease diagnostics, and therapeutic development.

DOI: https://doi.org/10.3791/68709
Nat Commun. 2025 Aug 11. 16(1): 7277

Computationally unmasking each fatty acyl C=C position in complex lipids by routine LC-MS/MS lipidomics.

Leonida M Lamp, Gosia M Murawska, Joseph P Argus, Aaron M Armando, Radu A Talmazan, Marlene Pühringer, Evelyn Rampler, Oswald Quehenberger, Edward A Dennis, Jürgen Hartler.

Identifying carbon-carbon double bond (C=C) positions in complex lipids is essential for elucidating physiological and pathological processes. Currently, this is impossible in high-throughput analyses of native lipids without specialized instrumentation that compromises ion yields. Here, we demonstrate automated, chain-specific identification of C=C positions in complex lipids based on the retention time derived from routine reverse-phase chromatography tandem mass spectrometry (RPLC-MS/MS). We introduce LC=CL, a computational solution that utilizes a comprehensive database capturing the elution profile of more than 2400 complex lipid species identified in RAW264.7 macrophages, including 1145 newly reported compounds. Using machine learning, LC=CL provides precise and automated C=C position assignments, adaptable to any suitable chromatographic condition. To illustrate the power of LC=CL, we re-evaluated previously published data and discovered new C=C position-dependent specificity of cytosolic phospholipase A2 (cPLA2). Accordingly, C=C position information is now readily accessible for large-scale high-throughput studies with any MS/MS instrumentation and ion activation method.

DOI: https://doi.org/10.1038/s41467-025-61911-x
Biomed Chromatogr. 2025 Sep;39(9): e70190

Development and Validation of a LC-MS/MS Method for Ripretinib and Its Metabolite: Example of a Journey From Laboratory Bench to Routine Application With a Greenness Assessment.

Cedric Rakotovao, Ahmad Sharanek, Audrey Burban, Paul Gueroue, Stéphane Bouchet, Guillaume Bouguéon, Dominique Ducint, Mathieu Molimard, Antoine Italiano, Sarah Djabarouti, Joris Guyon.

  Therapeutic drug monitoring of protein kinase inhibitors is widely practiced worldwide. Based on the example of ripretinib dosage requested by a clinician, we detailed the process of method development, using a literature-based approach while ensuring the sustainability of the method to be as environmentally friendly as possible. Therefore, a UPLC-MS/MS method for ripretinib and its active metabolite was optimized and validated using the corresponding stable isotopic internal standards in human plasma. The procedure employed a mobile phase mixture of water with 1% acetic acid and 0.1% formic acid, and acetonitrile. Positive electrospray ionization was performed, coupling with multiple reaction monitoring of m/z 510.4 → 417.4 and 510.4 → 389.4 for ripretinib, and 496.3 → 403.3 and 496.3 → 375.3 for N-desmethyl-ripretinib. The method was successfully validated according to the current version of the ICH Guideline provided by the EMA. The greenness assessment score of this procedure was better than previously published approaches using the AGREE metric. The validated UPLC-MS/MS method successfully monitored ripretinib and its metabolite concentrations in clinical and preclinical models.

Keywords:  LC‐MS/MS; N‐desmethyl‐ripretinib; TDM; pharmacokinetic; ripretinib

DOI:  https://doi.org/10.1002/bmc.70190
J Pharm Biomed Anal. 2025 Aug 06. pii: S0731-7085(25)00434-0. [Epub ahead of print]266 117093

Validation and clinical application of an LC-MS/MS method designed to simultaneously measure seven second-line TB drugs and two metabolites in human lung tissue.

Katie Kriegler Foster, Anil Pooran, Marthinus van der Merwe, Sandra Castel, Anton Joubert, Edda Zangenberg, Keertan Dheda, Lubbe Wiesner.

  We developed and validated a novel bioanalytical method for the simultaneous quantification of levofloxacin, linezolid, moxifloxacin, delamanid, bedaquiline, clofazimine, and pretomanid, along with the metabolites of delamanid (DM-6705) and bedaquiline (N-desmethyl-bedaquiline, M2), in human lung tissue samples. Following homogenization by bead beating and extraction by protein precipitation, the analytes were separated on an Agilent 1260 Infinity II HPLC system using a Poroshell 120 C18 EC (2.1 mm×50 mm, 2.7 µm) column with gradient elution, applying a mobile phase consisting of 0.1 % formic acid in water and 0.1 % formic acid in a mixture of acetonitrile and methanol. Detection and quantification of the analytes and their stable isotope labelled internal standards were performed on a Sciex API 5500 QTrap mass spectrometer using positive electrospray ionization and multiple reaction monitoring. Validation according to the guidelines of the FDA and EMA proved the method to be precise, accurate, and robust with no significant influence of matrix components. The application of the method to the analysis of clinical samples demonstrated the feasibility of quantifying the second-line anti-tuberculosis drugs in human lung tissue and the potential to provide insights into the drug distribution across the infection sites in the lung.

Keywords:  Lung tissue analysis; Method validation; Multiplex LC-MS/MS; Second-line TB drugs

DOI:  https://doi.org/10.1016/j.jpba.2025.117093
J Proteome Res. 2025 Aug 11.

Evaluating Novel Direct Injection Liquid Chromatography-Mass Spectrometry Method and Extraction-Based Workflows for Untargeted Lipidomics of Extracellular Vesicles.

Michał Młynarczyk, Felicja Gajdowska, Jorge Matinha-Cardoso, Paulo Oliveira, Paula Tamagnini, Mariusz Belka, Jagoda Mantej, Danuta Gutowska-Owsiak, Weronika Hewelt-Belka.

  Lipids of extracellular vesicles (EVs) are attracting attention due to their crucial biological functions and potential roles in processes such as carcinogenesis. This study compares three commonly used lipid extraction techniques, i.e., liquid-liquid extraction, single-phase extraction, and solid-phase extraction, with a novel direct injection liquid chromatography-mass spectrometry (DI-LC-MS) workflow tailored to EV lipidomics. In the DI-LC-MS approach, EVs are disrupted and released directly in the chromatographic system, enabling the analysis of lipids without a prior extraction step. The applicability of the DI-LC-MS workflow was demonstrated by profiling lipids in mammalian and bacterial EVs. The lipidome coverage and high precision of the DI-LC-MS method (coefficient of variation of peak area lower than 20% for all the identified lipids) enabled identification of differences in lipid profiles of EV samples. The column used in the DI-LC-MS method exhibited a sufficient lifespan and stability for comparative lipidomic studies. Lipidome coverage, lipid species distribution, and precision varied across the studied workflows; our findings highlight the strengths and limitations of these methods. The DI-LC-MS emerges as a sustainable alternative for EV lipidomic studies by eliminating the need for sample preparation and reducing analysis time, solvent use, and chemical noise while requiring less than 1 μL of sample.

Keywords:  LC-MS; extracellular vesicles; lipid extraction; lipidomics; liquid–liquid extraction; solid-phase extraction

DOI:  https://doi.org/10.1021/acs.jproteome.5c00156
Leg Med (Tokyo). 2025 Aug 07. pii: S1344-6223(25)00116-6. [Epub ahead of print]77 102682

Development of a simultaneous analysis method for 13 neonicotinoids and related insecticides in whole blood by LC-MS/MS using a Novum simplified liquid extraction column.

Tadashi Ogawa, Kyoko Maebashi, Tomohito Matsuo, Masae Iwai, Kana Sakamoto, Mamiko Fukuta, Katsutoshi Kubo, Fumio Kondo, Hiroshi Seno.

  We developed a simultaneous analysis method for 13 neonicotinoids and related insecticides in whole blood by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Novum simplified liquid extraction column. The extraction method, which employs an extraction solvent of 16% acetonitrile in ethyl acetate, demonstrated the best performance in terms of balancing phospholipid removal ability, recovery efficiency and matrix effects. Calibration curves showed good linear relationships, with r2 values exceeding 0.995. The intra- and inter-day accuracies and precisions were 85.5-108.8% and 0.2-13.0%, respectively. The recovery efficiencies were in the range of 52.7-92.7%. Matrix effects were between 60.6 and 201.0%; higher effects were seen for fipronil, which represents a significant challenge that resulted in substantial ion enhancement. Nevertheless, both the accuracy and precision of the method were enhanced by utilization of a stable isotopically-labeled internal standard followed by LC-MS/MS. The method was applied to a human postmortem blood sample collected during a forensic autopsy to demonstrate feasibility; results suggest that this method will be useful for forensic toxicological investigations.

Keywords:  Liquid chromatography–tandem mass spectrometry; Neonicotinoid; Protein and phospholipid removal column; Simplified liquid extraction column

DOI:  https://doi.org/10.1016/j.legalmed.2025.102682
Proteomics. 2025 Aug 13. e70026

Enhancing Lipidomics With High-Resolution Ion Mobility-Mass Spectrometry.

Gaoyuan Lu, Shuling Xu, Penghsuan Huang, Lingjun Li.

Lipids, indispensable yet structurally intricate biomolecules, serve as critical regulators of cellular function and disease progression. Conventional lipidomics, constrained by limited resolution for isomeric and low-abundance species, has been transformed by ion mobility-mass spectrometry (IM-MS). This technology augments analytical power through enhanced orthogonal separation, collision cross-section (CCS)-based identification, and improved sensitivity. This review examines the transformative advances in IM-MS-driven lipidomics, focusing on three major pillars: (1) a critical evaluation of leading ion mobility spectrometry (IMS) platforms, emphasizing innovative instrument geometries and breakthroughs in resolving lipid isomers; (2) an exploration of lipid CCS databases and predictive frameworks, spotlighting computational modeling and machine learning strategies that synergize experimental data with molecular representations for high-confidence lipid annotation; (3) emerging multi-dimensional lipidomics workflows integrating CCS with liquid chromatography-MS/MS to boost identification and depth, alongside mass spectrometry imaging for spatially resolved lipidomics. By unifying cutting-edge instrumentation, computational advances, and biological insights, this review outlines a roadmap for leveraging IM-MS to unravel lipidome complexity, catalyzing biomarker discovery and precision medicine innovation.

DOI: https://doi.org/10.1002/pmic.70026
Anal Methods. 2025 Aug 14.

Simple and robust analysis of cefuroxime in human plasma and bone tissues by LC-MS/MS.

Qixian Ling, Yuanyuan Zhang, Haotian Ma, Yanan Wang, Zhe Wang, Sihan Wang, Xin Xiong, Libo Zhao.

In end-stage osteoarthritis, total joint arthroplasty (TJA) represents the definitive therapeutic intervention. Cefuroxime, a second-generation cephalosporin, exhibits a broad spectrum of activity against both Gram-negative and Gram-positive microorganisms, making it a cornerstone of surgical antimicrobial prophylaxis (SAP) to mitigate prosthetic joint infection (PJI) risk. However, the escalating demand for revision arthroplasties has paralleled rising implant-associated infections, necessitating target-site pharmacokinetic optimization to ensure effective antibiotic exposure at the bone-implant interface. Therefore, we developed a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of cefuroxime in human plasma and bone tissues. The separation was completed in 7.5 min on a BEH C18 column (2.1 × 50 mm, 3.5 μm), and the gradient elution was performed in a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water at a flow rate of 0.3 mL min-1. The correlation coefficients of calibration curves were all greater than 0.99. The detection accuracy of plasma ranged from 93.11% to 98.60% (89.15-106.2% for bone). The intra- and inter-assay precision for both plasma and bone measurements were within 15% (20% at the lower limit of quantitation, LLOQ). The matrix effects were 2.34% to 2.91% in plasma and 3.13-5.17% in bone, while extraction recoveries ranged from 99.8% to 102.0% for plasma and 105.0-107.0% for bone. Upon stability assessment under varying storage conditions, all samples exhibited a difference of less than 15.0%. The method was successfully applied to the determination of cefuroxime in plasma and bone tissues of actual patients.

DOI: https://doi.org/10.1039/d5ay01085c
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 07. pii: S1570-0232(25)00304-6. [Epub ahead of print]1265 124750

Development and validation of a 'dilute and shoot' LC-MS/MS method in urine suitable for screening and confirmation analyses in a clinical setting.

Dimitra Florou, Amvrosios Orfanidis, Vassiliki A Boumba.

  Urine toxicological analysis serves as a significant tool in both clinical and forensic contexts, facilitating the diagnosis of acute intoxications, determination of causes of death, monitoring of substance use in occupational settings, and identification of drug-facilitated crimes. In this regard, the dilute-and-shoot method, when integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS), represents a promising analytical approach due to its efficacy, reliability, and wide-ranging applicability. This study presents the development and validation of an LC-MS/MS method that simultaneously detects and quantitates 115 drugs and metabolites comprising analytes from different categories, such as drugs of abuse (DOA), new psychoactive substances (NPS), prescription and over-the-counter drugs. Sample pretreatment was studied by applying different solvents (acetonitrile, water or methanol), and the best results were obtained after adding 100 μL of sample, 200 μL of a mixture of methanol: acetonitrile (3:1, v/v). The mixture was vortexed, centrifuged and the supernatant directly injected into the instrument. The analysis took place on a C18 column with a gradient elution over 7.5 min. The method was found to be selective and sensitive, offering LOD/LOQ ranging from 0.01 to 1.5/ 0.05-5 ng/mL, respectively. Validation of the method included evaluation of recovery, carryover, matrix effect, accuracy, precision, selectivity, stability and dilution integrity. The method performed satisfactorily and was therefore applied to urine samples that collected over a 12-week period from individuals enrolled in a rehabilitation program. The proposed method was applied as a follow-up tool aiming to detect prescribed (or not) medicine, as well as other illegal substances.

Keywords:  DOA; Dilute-and-shoot; LC-MS/MS; NPS; Screening; Urine; Validation

DOI:  https://doi.org/10.1016/j.jchromb.2025.124750
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 12. pii: S1570-0232(25)00317-4. [Epub ahead of print]1265 124763

Development and validation of simultaneous quantification of total and free clozapine and its two metabolites in human plasma using ultra high-performance liquid chromatography coupled to tandem mass spectrometry.

Rikako Kawanaka, Takahiro Sumimoto, Ryota Tanaka, Toshihiko Izumi, Moriaki Satoh, Masaaki Muronaga, Ryosuke Tatsuta, Hirofumi Hirakawa, Takeshi Terao, Hiroki Itoh.

  Several recent pharmacokinetic studies of clozapine (CLZ) and its metabolites have reported that plasma CLZ concentrations are associated with both efficacy and adverse effects, suggesting the usefulness of therapeutic drug monitoring. Although several quantification methods for total and free drug concentrations have been established, a simultaneous quantification method for total and free concentrations of CLZ and its metabolites using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has not been developed. In this study, we aimed to develop a simultaneous quantitative measurement method for wide ranges of plasma concentrations of total and free CLZ and its two major metabolites, N-desmethyl CLZ (NDC) and CLZ N-oxide (CNO), using UHPLC-MS/MS. Plasma samples were prepared by solid phase extraction, and the free fraction was obtained by ultrafiltration. This method meets the validation requirements of the U.S. Food and Drug Administration. The quantification method demonstrated good linearity over wide calibration ranges for total CLZ (10-1000 ng/mL), total NDC and CNO (40-4000 ng/mL), free CLZ (2-2000 ng/mL), and free NDC and CNO (0.8-800 ng/mL). Ultrafiltration recovery rates for free CLZ, NDC, and CNO were approximately 60.0 %, 65.4 %, and 72.8 %, respectively. The total drug recovery rates ranged from 91.4 % to 107.5 %, and the free drug recovery rates ranged from 88.5 % to 117.1 %. Furthermore, we successfully measured total and free CLZ, NDC, and CNO concentrations in plasma samples of 12 patients with schizophrenia treated with CLZ. We have successfully developed and validated a method for quantitative measurement of total and free CLZ, NDC, and CNO concentrations in plasma.

Keywords:  Clozapine; Clozapine N-oxide; N-desmethyl clozapine; Solid-phase extraction; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2025.124763
J Sep Sci. 2025 Aug;48(8): e70237

Measurement of Total and Free Cefiderocol Concentrations in Human Plasma by UHPLC-MS/MS: Application to Patients With Difficult-To-Treat Gram-Negative Bacterial Infections.

Raúl Rigo-Bonnin, Cristina El Haj, Eva Benavent, Judith Poblet-Florentin, Paula Pons-Oltra, Jaime Esteban, Oscar Murillo.

  Cefiderocol is an innovative siderophore cephalosporin against some of the most difficult-to-treat Gram-negative bacterial infections. As with other β-lactam antibiotics, therapeutic drug monitoring of cefiderocol in specific clinical scenarios could improve patient outcomes, minimize toxicity, and maximize treatment effectiveness. Thus, we aimed to develop and validate an ultra-high-performance liquid chromatography-tandem mass spectrometry procedure for measuring total and free cefiderocol concentrations in human plasma. Protein precipitation was used to extract patient samples. Equilibrium dialysis preceded sample preparation for measuring free cefiderocol concentrations. An Acquity-UPLC-BEH (2.1 mm × 100 mm id, 1.7 µm) column was selected for analyte separation, using 0.1% (v/v) formic acid in water and acetonitrile as mobile phases and working in a gradient containing. The cefiderocol and its internal standard ([2H8-cefiderocol) were detected by electrospray ionization mass spectrometry in positive and multiple reaction monitoring modes, using transitions of 752.2→285.3/468.2 and 760.2→293.3/468.2, respectively. Analysis time was 3.5 min per run. Precisions, absolute relative biases, normalized-matrix effect, and normalized recoveries were ≤13.9%, ≤14.2%, 95.7%-104.1%, and 95.9%-104.8%, respectively. Linearity was observed between 0.50 and 65.0 mg/L with linear regression coefficients greater than 0.9984. No significant interferences and carry-over were observed. The UHPLC-MS/MS procedures we present here could be suitable for clinical research purposes and hold promise for routine analysis. Their versatility would allow for application in the therapeutic drug monitoring of cefiderocol in subjects with difficult-to-treat Gram-negative bacterial infections to guide the treatment and adjust the daily dose of this new β-lactam antibiotic.

Keywords:  UHPLC‐MS/MS; cefiderocol; free drug; rapid equilibrium dialysis; therapeutic drug monitoring; β‐lactam antibiotics

DOI:  https://doi.org/10.1002/jssc.70237
J Chromatogr A. 2025 Aug 05. pii: S0021-9673(25)00606-5. [Epub ahead of print]1760 466261

Analysis of per-and polyfluorinated alkyl substances by chromatography-mass spectrometry: A review.

Peng Liu, Jiaoyang Li, Liangyi Hong, Huimin Zou, Xingyan Zhou, Xin Zhang, Yan Zhou, Hian Kee Lee, Zhenzhen Huang.

  Per- and polyfluoroalkyl substances (PFAS), a group of synthetic chemicals that have been produced for over fifty years, are of significant concern due to their widespread environmental distribution and potential risks to human health. The transition from legacy PFAS to emerging PFAS with shorter chains and unknown structures has further amplified existing challenges in their analysis. A variety of reliable and sensitive analytical methods is available to analyze these contaminants in various biological and environmental matrices. However, current research on PFAS lacks a unified standard for their determination and there is limited guidance on selecting appropriate methods to address specific analytical challenges. In this review, up-to-date analytical methods for PFAS detection based on chromatography and mass spectrometry in the recent literature are reviewed. The survey covers (i) a discussion of the advantages, limitations, and scope of application of various sample preparation methods for PFAS analysis, (ii) recent advances in chromatographic separation and mass spectrometry with respect to targeted and non-targeted analysis, and (iii) progress in the analysis of PFAS in complex sample matrices, including a wide range of environmental and biological samples. While liquid chromatography-mass spectrometry has been the mainstay technique in most of the studies covered, greater efforts are needed to develop newer methods to deal with not only the legacy compounds but also emerging ones. A wide range of biological and environmental matrices also needs to be identified and considered in future analytical and sample preparation developmental work.

Keywords:  Chromatography; Mass spectrometry; Per- and polyfluoroalkyl substances; Sample preparation

DOI:  https://doi.org/10.1016/j.chroma.2025.466261
Diagnostics (Basel). 2025 Jul 25. pii: 1870. [Epub ahead of print]15(15):

Development of a Liquid Chromatography-Tandem Mass Spectrometry Method for Oxylipin Analysis and Its Application to Children's Plasma.

Yonghan Li, Siddabasave Gowda B Gowda, Divyavani Gowda, Atsuko Ikeda, Yu Ait Bamai, Rahel Mesfin Ketema, Reiko Kishi, Hitoshi Chiba, Shu-Ping Hui.

  Background/Objectives: Oxylipins, a family of oxygenated natural products derived from polyunsaturated fatty acids (PUFAs), play crucial roles in various physiological processes. Evaluating their levels in vivo helps to reveal their roles in health and disease. Because of the numerous isomers of oxylipins, it is essential to develop efficient and precise analytical methods for their identification and quantification. The objective of this study is to establish a quantitative method for oxylipin analysis and its application to the assessment of oxylipins in children's plasma, with potential implications for diagnostic use in pediatric populations. Methods: A liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed to quantify 64 oxylipins and four precursor PUFAs within 36 min. The limits of quantification ranged from 0.25 to 50 pg, with most analytes showing recoveries and matrix effects between 85 and 110% and between 90 and 110%, respectively. Intra- and inter-day precision values were within 15%. The established method was applied to plasma samples from children aged 9-12 years (boys = 181; girls = 161) in Hokkaido, Japan, to assess the relation between plasma oxylipin and PUFA levels and age, sex, and body mass index. Results: There was no significant correlation between oxylipin levels and age, sex, or body mass index. However, among the PUFAs, boys had higher eicosapentaenoic acid and arachidonic acid levels than those of girls, with a significant increase in eicosapentaenoic acid levels in the overweight group compared with those in the underweight group. Conclusions: We successfully developed a simple and highly selective method for the analysis of oxylipins in preadolescent children's plasma samples. Thus, this study provides a foundation for broader application of the developed method to different biological samples in future studies.

Keywords:  children; liquid chromatography; mass spectrometry; oxylipins; plasma; polyunsaturated fatty acids

DOI:  https://doi.org/10.3390/diagnostics15151870
Bioanalysis. 2025 Aug 14. 1-9

Ultra-high throughput mass spectrometry with ultra high-speed separations: differential mobility spectrometry-acoustic ejection mass spectrometry (DAEMS).

Samad Bazargan, Chang Liu, Bradley B Schneider, Thomas R Covey.

  With the ongoing advancements in the field of ambient ionization for mass spectrometry, systems with a high-throughput capability on the order of 1 sample/second are readily available. This has led to the adoption of mass spectrometry for a wide variety of applications including those in the drug discovery process. Mass spectrometers have traditionally relied on pre-separation technologies such as high-pressure liquid chromatography for sample clean-up and isobaric separations, but such techniques are not high-throughput compatible. Differential mobility spectrometry is a high-speed atmospheric separation device with separations orthogonal to m/z that can be coupled with the high-throughput sample introduction devices such as acoustic ejection mass spectrometer to address this gap. In this article we highlight the significance of the recent reports on this topic and provide some insights into expanding the use of this technique for new applications. We believe this is a promising new development and will help propel the high-throughput mass spectrometry beyond isobaric interferences.

Keywords:  High-throughput analysis; acoustic ejection mass spectrometry; differential mobility spectrometry; drugs of abuse; high throughput screening; isomer separation

DOI:  https://doi.org/10.1080/17576180.2025.2546780
PLoS One. 2025 ;20(8): e0324668

An automated software-assisted approach for exploring metabolic susceptibility and degradation products in macromolecules using high-resolution mass spectrometry.

Paula Cifuentes, Ismael Zamora, Tatiana Radchenko, Fabien Fontaine, Albert Garriga, Luca Morettoni, Jesper Kammersgaard Christensen, Hans Helleberg, Bridget A Becker.

A comprehensive understanding of drug metabolism is crucial for advancements in drug development. Automation has improved various stages of this process, from compound procurement to data analysis, but significant challenges persist in the metabolite identification (MetID) of macromolecules due to their size, structural complexity, and associated computational demands. This study introduces new algorithms for automated Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS) data analysis applicable to macromolecules. A novel peak detection approach based on the most abundant mass (MaM) is presented and systematically compared with the monoisotopic mass (MiM) approach, commonly used in small molecules MetID. Additionally, three structure visualization strategies, expanded (atom-level), non-expanded (monomer-level), and a hybrid mode, are evaluated for their impact on computation data processing time and interpretability, based on their distinct fragmentation strategies. The workflow was validated using six diverse datasets, comprising linear and cyclic peptides and oligonucleotides with both natural and unnatural monomers, covering a molecular weight range of 700-7630 Da. A total of 970 metabolites were identified under various experimental and ionization conditions. The MaM algorithm demonstrated higher scores and a greater number of matches, instilling greater confidence in the accurate prediction of metabolite structures, while the non-expanded visualization significantly reduced processing times (ranging from minutes to under an hour for most peptides). Furthermore, the visualization algorithm, which integrates monomer-level and atom/bond notation, enables clear localization of metabolic biotransformations. Compared to previous studies, the proposed workflow demonstrated reduced processing time, consistent detection of degradation products, and enhanced visualization capabilities, advancing automated MetID for macromolecules.

DOI: https://doi.org/10.1371/journal.pone.0324668
IEEE Trans Comput Biol Bioinform. 2025 Jul-Aug;22(4):22(4): 1629-1640

MetaboliticsDB: A Database of Metabolomics Analyses.

M Hasan Celik, Onurcan Ersen, Taj Saleh, Alper Dokay, Ahmet Enis Guven, Ali Cakmak.

Web-based metabolomics databases store relative metabolite abundance datasets measured under different physiological conditions. However, their pathway-level analysis capabilities are mostly limited to superimposing the measurements onto the pathways of the measured metabolites. Besides, none of the existing metabolomics databases offer tools to store, manage, compare, and search metabolomics analysis results. In this paper, we present MetaboliticsDB, which features a database of metabolomics analyses and a set of associated analytics tools. It enables users to store and compare their metabolomics analysis results against others to study, for instance, the progression of a disease. Moreover, MetaboliticsDB implements a genome-scale metabolic network-based analysis tool (i.e., Metabolitics) that performs network-based flux analysis. Besides, MetaboliticsDB features an advanced querying interface offering flexible criteria, such as listing all analyses where a certain pathway experiences a major increase in activity, to help researchers identify conditions sharing a similar mechanism. Finally, MetaboliticsDB employs AI-based models to associate the studied metabolomics data with diseases. Currently, the database contains analysis results for 2,174 individuals and 40 diseases. We demonstrate MetaboliticsDB's usage with a case study on Hepatocellular Carcinoma. Our experimental evaluation shows that MetaboliticsDB provides biologically relevant metabolic network-level analysis results, disease association with high accuracy, and a scalable architecture.

DOI: https://doi.org/10.1109/TCBBIO.2025.3563807
J Chromatogr A. 2025 Jul 18. pii: S0021-9673(25)00563-1. [Epub ahead of print]1760 466217

Development of a sensitive UPLC-MS/MS method for simultaneous determination of 15 Bisphenol A analogues in human serum and urine: Application of paired samples and global exposure assessment.

Guangting Hu, Zhichun Zhang, Yi Huang, Deyu Fang, Ming Zhan, Weidong Qu, Ying Zhou.

  The global replacement of Bisphenol A (BPA) with structurally similar yet understudied analogues demands precise biomonitoring tools to assess human exposure and health risks. To bridge critical gaps, we developed a novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 15 emerging BPA analogues including Bisphenol AF (BPAF), Bisphenol AP (BPAP), Bisphenol B (BPB), Bisphenol C (BPC), Bisphenol E (BPE), Bisphenol F (BPF), Bisphenol G (BPG), Bisphenol FL (BPFL), Bisphenol M (BPM), Bisphenol P (BPP), Bisphenol pH (BPPH), Bisphenol S (BPS), Bisphenol TMC (BPTMC), Bisphenol Z (BPZ), and Bisphenol A diglycidyl ether (BFDGE)-in human serum and urine matrices. The method achieved ultrahigh sensitivity with limits of detection (LODs) of 0.008-0.032 μg/L in urine and 0.010-0.030 μg/L in serum, high accuracy (recoveries: 84.58-113.53 %), and reproducibility (relative standard deviation (RSD) ≤14.7 %). Rigorous validation was conducted through uncertainty assessment and green chemistry metrics (GAC) evaluation. The following scores were obtained: Analytical GREEnness metric approach (AGREE):0.65, Analytical GREEnness Metric for Sample Preparation (AGREEprep):0.64, Modified Green analytical procedure index (MoGAPI) :72, Complex modified Green analytical procedure index (ComplexMoGAPI):73, RGB model: 74.2 %, Blue Applicability Grade Index (BAGI):60, Click Analytical Chemistry Index (CACI):69 and Carbon Footprint Reduction Index (CaFRI):71, respectively. These results confirm the method's reliability and environmental sustainability. Applied to 44 paired samples from Chinese children, the study revealed serum biomarkers (BPPH + BPTMC) as superior indicators of internal exposure compared to urinary metabolites, providing the first-reported paired biospecimen data for these analogues. Furthermore, dual-matrix biomonitoring (serum + urine) significantly improved cumulative exposure assessment accuracy. This work establishes a robust analytical framework for tracking emerging BPA substitutes, supporting evidence-based chemical regulation and advancing environmental health research.

Keywords:  Biomonitoring; Bisphenols; Global exposure assessment; Paired samples; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2025.466217
Plants (Basel). 2025 Jul 24. pii: 2284. [Epub ahead of print]14(15):

The Role of LC-MS in Profiling Bioactive Compounds from Plant Waste for Cosmetic Applications: A General Overview.

Gilda D'Urso, Alessandra Capuano, Francesca Fantasma, Maria Giovanna Chini, Vincenzo De Felice, Gabriella Saviano, Gianluigi Lauro, Agostino Casapullo, Giuseppe Bifulco, Maria Iorizzi.

  The agro-industrial sector produces large amounts of by-products that have a high environmental impact, so it has become essential to recover food waste at all levels. This is because it often contains bioactive molecules that can be a valuable source of new products such as animal feed, biopolymers, or products for human use, (e.g., cosmetics and nutraceuticals) due to its antioxidant, antimicrobial, and anti-inflammatory properties. Advanced analytical methodologies such as liquid chromatography coupled to mass spectrometry (LC-MS) are crucial for the characterisation of bioactive chemicals in these waste materials. LC-MS enables both targeted and untargeted metabolomic approaches, facilitating the identification and quantification of a wide range of secondary metabolites, including polyphenols, flavonoids, alkaloids, and terpenoids. The choice of extraction methodology is essential for the precise identification and quantification of these metabolites. This study provides an overview of LC-MS as an effective tool for analysing complex extracts derived from plant waste, discussing both methodological aspects and typical bioactive metabolites identified, and offering examples of their potential applications in cosmeceutics.

Keywords:  LC-MS analysis; bioactive compounds; cosmetic applications; metabolomics; plant waste

DOI:  https://doi.org/10.3390/plants14152284
Food Res Int. 2025 Oct;pii: S0963-9969(25)01248-7. [Epub ahead of print]218 116910

Quantitation of amylase/trypsin-inhibitors in barley using targeted LC-MS/MS.

Sarah Joestl, Dalia Z Alomari, Ahmad M Alqudah, Andreas Börner, Sabrina Geisslitz, Katharina A Scherf.

  Amylase/trypsin-inhibitors (ATIs) are known allergens and triggers of non-celiac wheat sensitivity. Until now, ATIs were only quantitated in wheat species. We developed and validated a targeted stable isotope dilution analysis LC-MS/MS method to quantitate ten barley-specific ATIs, including one monomeric and one dimeric amylase-inhibitor, four chloroform/methanol-soluble types, three subtilisin/chymotrypsin-inhibitors and one amylase/subtilisin-inhibitor. After successful validation in terms of precision, recovery and limits of detection and quantitation, the method was applied to 181 barley accessions from the Global EcoSeed panel, comprising 113 two-row and 68 six-row barleys of different genetic backgrounds. The overall ATI content was 1.1-5.2 mg/g, corresponding to 0.7-3.6 % of the total protein content with no clear distinction between two-row and six-row barleys. This study is the first to provide insights on the ATI content and composition of barley, which can be used to make low-ATI foods for special dietary needs.

Keywords:  Allergen; LC-MS/MS; Non-celiac gluten sensitivity (NCGS); Non-celiac wheat sensitivity (NCWS); Stable isotope dilution analysis

DOI:  https://doi.org/10.1016/j.foodres.2025.116910
J Am Soc Mass Spectrom. 2025 Aug 12.

Long-Term System Suitability Evaluation for Mass Accuracy in the Analysis of Small Molecules by High-Resolution Mass Spectrometry.

Paul Löffler, Svante Rehnstam, Lutz Ahrens, Foon Yin Lai, Alberto Celma.

  High-resolution mass spectrometry (HRMS) is critical for the identification and characterization of (un)known organic chemicals. In this regard, ensuring high mass accuracy in HRMS instruments is essential for reliable results in nontarget and suspect screening. This study presents a practical approach for evaluating and maintaining mass accuracy over time using ultrahigh pressure liquid chromatography coupled with electrospray ionization Orbitrap HRMS. A set of 13 reference standards, encompassing a range of polarities and chemical families, was analyzed before and after sample analysis batches to assess the impact of various factors on the instrumental performance regarding mass accuracy. The aim is not to recalibrate the system but to provide a reliable snapshot of the mass accuracy over time. The study found that the positive ionization mode exhibited higher accuracy and precision compared with the negative mode. Factors affecting mass accuracy included calibration quality, the number of batch injections, and the time between calibrations, where the two latter factors were related to each other. Results suggest that performing system suitability tests for high-resolution accurate masses with two injections before and after sample analysis is adequate for ensuring acceptable mass spectrometric performance for robust and reliable HRMS data acquisition, but performing three injections is recommended. This protocol ensures that informed decisions can be made with regard to the mass accuracy, the calibration, and a potential recalibration before HRMS data acquisition is performed.

Keywords:  Orbitrap; instrumental performance; nontarget screening; quality assurance; reproducibility; suspect screening

DOI:  https://doi.org/10.1021/jasms.5c00128