bims-metlip 2026-01-04 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–01–04
23 papers selected by
Sofia Costa, Matterworks

Simultaneous quantification of acetylsalicylic acid, clopidogrel, ticagrelor and their major metabolites in human plasma by liquid chromatography-tandem mass spectrometry.
Development and Validation of a Simultaneous Quantification Method for 12 Targeted Therapies and 3 Active Metabolites in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry: An Application to Therapeutic Drug Monitoring in Patients With Hematological Malignancies.
Development and Application of Analytical Method for Simultaneous Quantification of Iopromide and Iodixanol in Human Plasma.
Determination of N-Lactoyl-phenylalanine, phenylacetylglutamine and trimethylamine-N-oxide related metabolites in hypertensive patients by liquid chromatography-mass spectrometry and the application to blood pressure regulation.
Robust Workflow for Multiclass Host-Gut Microbial Cometabolite Quantitation in Human Stool via 3-Nitrophenylhydrazine Derivatization and LC-MS/MS: A Validated Analytical Platform for Translational Studies.
Development and Validation of an LC-MS/MS Method for the Quantification of Methenamine in Raw Milk and Bovine Muscle and Its Application to Incurred Samples.
Distinguishing 4- vs 5-Hydroxy-N,N-Dimethyltryptamine (Psilocin vs Bufotenine) Using Hydrogen-Deuterium Back-Exchange.
Rapid, robust LC-MS/MS quantification of cefazolin in whole blood microsamples, plasma, and plasma ultrafiltrate: Analytical method validation and preliminary clinical application in pediatric population.
Improved preanalytical workflow for pancreatic tissue lipidomics: Insights into lipid stability and polar lipid recovery.
A Protocol for Robust Discovery Proteomics Using Nano Liquid Chromatography Using Pillar-Array Column Technology and High-Resolution Mass Spectrometry With Data-Independent Acquisition.
spatialstein: An Open-Source Workflow for Annotation, Deconvolution, and Spatially Aware Segmentation of Mass Spectrometry Imaging Data.
Spatial Mapping of PUFA Incorporation into Phospholipids with bisallylic deuteration.
An ID-HPLC-MS/MS based candidate reference measurement procedure for the quantification of imipenem, meropenem and ertapenem in human plasma.
Coupling High-Throughput Magnetic-Bead-Based Immunoaffinity with LC-MS/MS Analysis for Quantifying Cylindrospermopsin in Different Fish Tissues.
Filtration-Based Rapid ECM-Hydrogel Elimination Enables Robust MALDI MSI of Intestinal Organoids.
Urinary metabolome dynamics in 13C-labeled mice.
Mass Spectrometry Imaging Elucidates the Precise Localization and Site-Specific Functions of Skin Lipids.
Determination of 15 Restricted Substances in Plastic-Packaged Food by Gas Chromatography-Mass Spectrometry.
Temperature-Responsive Liquid Chromatography for One- and Two-Dimensional Separations in Compound-Specific Isotope Analysis.
MetImputBERT: a pretrained BERT framework for missing value imputation in NMR metabolomics data.
Development and Validation of an Assay Method for Levothyroxine Soft Gelatin Capsules Utilizing the RP-HPLC Technique.
An Interlaboratory Comparison of High-Resolution Ion Mobility Collision Cross-Sections from Human Plasma.
Desorption Electrospray Ionization Mass Spectrometry Imaging: Principles, Advancements, and Multidisciplinary Applications.

Clin Chim Acta. 2025 Dec 31. pii: S0009-8981(25)00695-3. [Epub ahead of print] 120816

Simultaneous quantification of acetylsalicylic acid, clopidogrel, ticagrelor and their major metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

Wenqiang Wang, Lijiao Zhang, Ciren Baima, Shenghao Gao, Xiaoyu Sun, Xiaoguang Xu, Yunfeng Cao.

  In recent years, there has been a critical need for therapeutic drug monitoring (TDM) of antiplatelet agents. To address this, we developed a sensitive, rapid, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as a unified assay for the simultaneous quantification of aspirin (ASA), salicylic acid (SA), clopidogrel (CLP), its carboxylic acid metabolite (CLPM), ticagrelor (TGL), and its active metabolite AR-C124910XX for the first time. Plasma samples were pretreated by protein precipitation and separated on an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) with a total run time of 4 min. Mass spectrometry was performed in polarity-switching mode, and quantification was achieved using multiple reaction monitoring (MRM) with stable isotope-labeled internal standards (SA-d6, CLPM-d4, TGL-d7) for normalization. Method validation demonstrated excellent linearity over the range of 0.5-2000 ng/mL (R2 ≥ 0.9938), with extraction recoveries of 98.2 %-107 % and negligible matrix effects. Stability testing indicated that all analytes were generally stable under clinically relevant conditions; however, ASA underwent rapid esterase-mediated hydrolysis at room temperature, forming SA as the major degradation product, whereas it remained relatively stable at 2-8 °C and was stable for at least 15 days at -80 °C. The method was successfully applied to plasma samples from 61 cardiovascular patients, confirming its utility for evaluating medication adherence as well as inter- and intra-individual variability in drug exposure. In conclusion, the developed LC-MS/MS method provides high sensitivity, high throughput, and robust performance, offering a powerful analytical tool to support individualized antiplatelet therapy.

Keywords:  Acetylsalicylic acid; Clopidogrel; LC–MS/MS; Pharmacokinetics; Therapeutic drug monitoring; Ticagrelor

DOI:  https://doi.org/10.1016/j.cca.2025.120816
Ther Drug Monit. 2025 Dec 30.

Development and Validation of a Simultaneous Quantification Method for 12 Targeted Therapies and 3 Active Metabolites in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry: An Application to Therapeutic Drug Monitoring in Patients With Hematological Malignancies.

Nicolas Vignal, Marie Allard, Sofiane Fodil, Etienne Lengline, Hélène Sauvageon, Marie Sébert, Alain Plé, Delphine Rea, Samia Mourah, Lauriane Goldwirt.

   BACKGROUND: Treatment strategies for hematological cancers and immunological diseases increasingly incorporate targeted oral therapies. These drugs provide improved quality of life but exhibit complex pharmacokinetics. Therapeutic drug monitoring (TDM) may help optimize treatment in various clinical situations, including managing drug-drug interactions, assessing adherence, evaluating exposure-response relationships, and investigating suspected drug toxicities. In this study, we developed and validated a sensitive liquid chromatography-tandem mass spectrometry method for simultaneous quantification of multiple targeted therapies and applied this method to clinical samples for TDM.
METHODS: After simple protein precipitation of plasma samples, chromatographic separation was performed on a UPLC system coupled with MS/MS in positive ionization mode. The mobile phase consisted of a gradient elution using 10 mM of ammonium formate with 0.1% (v/v) formic acid (phase A) and acetonitrile with 0.1% (v/v) formic acid (phase B), at a flow rate of 300 µL/min.
RESULTS: The analysis time was 7.0 minutes per run. Calibration curves were linear over the ranges of 0.5-500 ng/mL for ruxolitinib, tofacitinib, baricitinib, and hydroxyquizartinib AC488; 5-2500 ng/mL for asciminib, gilteritinib, and quizartinib; 50-10000 ng/mL for ivosidenib, venetoclax, midostaurin, CGP52421, CGP62221, and pacritinib; 100-100000 ng/mL for enasidenib; and 500-100000 ng/mL for eltrombopag. All analytes showed correlation coefficients above 0.99. Intra- and interday precision values were below 14.67%.
CONCLUSIONS: We developed and validated a sensitive liquid chromatography-tandem mass spectrometry method requiring only 50 µL of plasma volume for the quantification of 12 targeted oral anticancer drugs and 3 active metabolites. This multianalyte assay offers strong potential for TDM in patients receiving contemporary anticancer treatments.

Keywords:  asciminib; gilteritinib; ivosidenib; quizartinib; venetoclax

DOI:  https://doi.org/10.1097/FTD.0000000000001426
Int J Anal Chem. 2025 ;2025 2432521

Development and Application of Analytical Method for Simultaneous Quantification of Iopromide and Iodixanol in Human Plasma.

Liya Ye, Nianxi Yu, Peng Huan, Zhengyan Liang, Zhenhui Jiang, Xinran Wang, Zhipeng Wang, Xia Tao, Shouhong Gao.

  The purpose of this study is to develop and validate a simple and rapid analytical method using ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous quantification of two commonly used contrast agents, iopromide and iodixanol, in human plasma. The separation of two compounds was analyzed utilizing an Agilent ZORBAX SB-C18 column (2.1 × 100 mm, 3.5 μm) with an isocratic elution procedure. The mobile phase consisted of acetonitrile and 0.2% formic acid aqueous solution (14:86, V:V). A simple protein precipitation method was used to pretreat plasma samples. The iopromide and iodixanol exhibited excellent linearity between 2.0 and 400.0 μg/mL, with both R values exceeding 0.99. Recovery of iopromide ranged from 91.39% to 102.69%, and matrix effect varied between 87.88% and 104.08%; the relative standard deviation (RSD%) of intra- and interday precisions fell within the range of 1.29%-4.7%. For iodixanol, recovery ranged from 97.68% to 100.14%, and the matrix effect was between 87.88% and 96.64%, and RSD% values of intra- and interday precisions ranged from 1.58% to 8.2%. Method validation results all met methodological criteria. The UHPLC-MS/MS method was successfully developed and validated and then applied to determine two common contrast agents, iopromide and iodixanol, in human plasma.

Keywords:  UHPLC-MS/MS; iodixanol; iopromide; therapeutic drug monitoring

DOI:  https://doi.org/10.1155/ianc/2432521
J Pharm Biomed Anal. 2025 Dec 29. pii: S0731-7085(25)00668-5. [Epub ahead of print]271 117327

Determination of N-Lactoyl-phenylalanine, phenylacetylglutamine and trimethylamine-N-oxide related metabolites in hypertensive patients by liquid chromatography-mass spectrometry and the application to blood pressure regulation.

Xin Tan, Xianyun Jiang, Lu Ren, Ziyu Meng, Jingyi Yang, Yuan Wang.

  Metabolites, such as N-lactoyl-phenylalanine (Lac-Phe), phenylacetylglutamine, and trimethylamine-N-oxide related precursors, may be associated with hypertension. A rapid, sensitive and accurate method, with high throughput, low sample volume, and easy operation to analyze these metabolites would facilitate their measurement and provide important insights into their role in hypertension. We developed an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous measurement of eight metabolites in 5 µL human plasma samples with a 4.2-min chromatographic run and easy operation. The method achieved good linearity (R² > 0.99), sensitivity (limit of quantification: 0.1-1 ng/mL), accuracy (within-day: -5.33 % to 11.38 %; between-day: -3.35 % to 11.50 %), precision (within-day: 1.35-13.25 %; between-day: 1.74-12.73 %), matrix effect (-5.93 % to 14.81 %), recovery rates (91.49 % to 103.31 %), bench-top stability (coefficient of variations [CVs]: 1.02-6.16 %), long-term stability (CVs: 3.63-12.94 %), on-instrument stability (CVs: 0.53-5.09 %) and freeze-thaw stability (biases: -12.60 % to -0.13 %). We successfully utilized the UHPLC-MS/MS method to measure plasma samples obtained from 204 healthy individuals and 204 patients with hypertension. Plasma Lac-Phe was reduced in patients with hypertension, and the potential effects of blood pressure regulation by Lac-Phe were explored. In angiotensin II-induced hypertensive mice, Lac-Phe alleviated the increases in systolic and diastolic blood pressure. These findings highlight the therapeutic potential of Lac-Phe in hypertension.

Keywords:  Blood pressure regulation; Healthy individuals; Hypertension; Lac-Phe; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2025.117327
Anal Chem. 2025 Dec 29.

Robust Workflow for Multiclass Host-Gut Microbial Cometabolite Quantitation in Human Stool via 3-Nitrophenylhydrazine Derivatization and LC-MS/MS: A Validated Analytical Platform for Translational Studies.

Daniel Zhi Wei Ng, Mingyou Koh, Adrian Low, Lin Liu, Nur Halisah Binte Jumat, Zixuan Zhang, Xiu Qi Koh, Mengtong Zhu, Mark Muthiah, Yock Young Dan, Jonathan Wei Jie Lee, Eric Chun Yong Chan.

Host-gut microbial co-metabolites, including short-chain fatty acids (SCFA), bile acids (BA), tryptophan metabolites, and branched-chain amino acids (BCAA), have key immune-metabolic functions affecting human health. Dysbiosis-induced alterations in their levels are implicated in the pathogenesis of diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). However, simultaneous quantitation of these chemically diverse analytes in stool remains analytically challenging due to their diverse physicochemical properties and wide concentration ranges. Here, we developed and rigorously validated a derivatization and targeted liquid chromatography tandem mass spectrometry workflow for the simultaneous quantitation of host-gut microbial cometabolites in human stool. A 3-nitrophenylhydrazine derivatization protocol was optimized by systematically adjusting reagent concentrations and introducing postreaction quenching to suppress in-line acetic acid derivatization. Chromatographic separation was enhanced by using a novel dual-additive mobile-phase strategy (formic acid and ammonium acetate in aqueous and organic phase, respectively) coupled to a mixed-mode C18-anion-exchange stationary phase, enabling improved resolution and sensitivity across chemically diverse metabolite classes. Our optimized analytical method achieved accurate, sensitive, and efficient quantitation of 38 metabolites (15 SCFA, 16 BA, 4 tryptophan metabolites, 3 BCAA) within 23 min, demonstrating excellent linearity (r2 > 0.99) and precision (CV < 15%), with short- (autosampler, 4 °C) and long-term (freezer, -20 °C) stability. Comparative analysis of healthy controls and MASLD stools revealed distinct metabolic signatures, including reduced SCFA and C6-oxidized BA, and elevated conjugated and secondary BA derivatives in MASLD. Our study establishes an analytically rigorous platform for multiclass host-gut cometabolite quantitation in stool, with demonstrated utility for translational research into gut-liver axis disorders.

DOI: https://doi.org/10.1021/acs.analchem.5c05360
Molecules. 2025 Dec 17. pii: 4807. [Epub ahead of print]30(24):

Development and Validation of an LC-MS/MS Method for the Quantification of Methenamine in Raw Milk and Bovine Muscle and Its Application to Incurred Samples.

Sunjin Park, Chung-Oui Hong, Se-Hyung Kim, Seon-Young Lee, Inhae Jeon, Do Hui Kim, Hyun-Ok Ku, Mi-Young Park.

  Methenamine, a urinary antiseptic with antimicrobial properties, decomposes into toxic formaldehyde under acidic conditions. Its use is prohibited in dairy cattle in Korea to prevent harmful residues in milk. This study was designed to develop and validate a sensitive and reliable LC-MS/MS method for determining methenamine in raw milk and bovine muscle in compliance with the Positive List System (PLS) regulations. Samples were extracted with acetonitrile (ACN)-methanol (MeOH) (7:3, v/v) containing ammonia water, followed by defatting with n-hexane and purification with primary secondary amine (PSA). Chromatographic separation was performed on a hydrophilic interaction liquid chromatography (HILIC) column, and quantification was conducted using matrix-matched calibration to minimize matrix effects. The method showed excellent linearity (R2 > 0.999), low limits of quantification (LOQ) (0.49 μg/kg for raw milk; 0.64 μg/kg for bovine muscle), and acceptable recoveries (78.1-102.8%) with precision (CV ≤ 8.75%), meeting Codex CAC/GL 71-2009 criteria. Stability studies demonstrated that methenamine remained stable in stock solutions, working standards and processed extracts under the storage and handling conditions used. Application to incurred samples resulted in the detection of methenamine in 2 of 32 raw milk samples (0.65 and 1.14 μg/kg) but in none of the 25 bovine muscle samples, with all detected levels below the Korean PLS limit. These findings confirm that the developed method is accurate, sensitive, and applicable for routine surveillance of methenamine residues to ensure consumer safety.

Keywords:  LC–MS/MS; Positive List System (PLS); bovine muscle; methenamine; raw milk; residue analysis

DOI:  https://doi.org/10.3390/molecules30244807
J Am Soc Mass Spectrom. 2025 Dec 30.

Distinguishing 4- vs 5-Hydroxy-N,N-Dimethyltryptamine (Psilocin vs Bufotenine) Using Hydrogen-Deuterium Back-Exchange.

Michael W Christopher, Boone M Prentice, Timothy J Garrett.

  Distinguishing metabolite isomers often relies on comparing relative data, such as relative chromatographic retention times and ion mobility arrival time orders, or relative product ion abundances. These approaches necessitate the need for quality reference data and/or chemical standards. An ideal method for differentiating isomers would leverage one of the absolute physiochemical properties of the isomers, and would have no reliance on instrument vendor, chromatographic column chemistry, or external reference data. For example, the pKa of an aromatic hydroxy hydrogen changes according to ring position across isomers (e.g., 4- vs 5-hydroxyindole). Herein, we leverage the difference in pKa to resolve 4- and 5-hydroxy positional isomers of hydroxy-N,N-dimethyltryptamine (psilocin and bufotenine), the structural moiety of compounds with profound effects on the serotonergic system. We first use hydrogen-deuterium exchange (HDX) to rapidly exchange the indole amine hydrogen and gradually exchange the indole hydroxy hydrogen atoms to deuterium atoms. We then back-exchange the indole amine deuterium atom back to a hydrogen atom on the LC column and monitor the kinetic exchange rates of the retained aromatic hydroxy deuterium atom using high resolution mass spectrometry (HRMS). HDX kinetics allow for facile differentiation of the two isomers, with only 4-hydroxy-N,N-dimethyltryptamine exchanging at an appreciable amount within hours. These results could ultimately be used to characterize a variety of unknown structural isomers.

Keywords:  , indole; HDX; high-resolution mass spectrometry; isomers; liquid chromatography; metabolomics; tautomers

DOI:  https://doi.org/10.1021/jasms.5c00421
Clin Chim Acta. 2025 Dec 25. pii: S0009-8981(25)00687-4. [Epub ahead of print]582 120808

Rapid, robust LC-MS/MS quantification of cefazolin in whole blood microsamples, plasma, and plasma ultrafiltrate: Analytical method validation and preliminary clinical application in pediatric population.

Arkadiusz Kocur, Agnieszka Czajkowska, Mateusz Moczulski, Anna Częczek, Małgorzata Mikaszewska-Sokolewicz.

  Optimal dosing of cephalosporins in pediatric ICU patients is difficult due to high inter-individual variability in β-lactam pharmacokinetics, limited blood volume, and the lack of validated microsampling-compatible clinical assays. We developed and validated a rapid and reliable liquid chromatography-tandem mass spectrometry method to measure cefazolin concentrations in lithium-heparin plasma, plasma ultrafiltrate (unbound fraction), and capillary whole blood collected with volumetric absorptive microsampling (VAMS). This method was applied in a pilot cohort of pediatric patients. Samples were protein precipitated with cryo-cooled organic solvents, separated by reversed-phase liquid chromatography (reversed-phase HPLC) using a polar C18 column, and detected by multiple reaction monitoring (MRM) on a hybrid triple quadrupole-linear ion trap (QTRAP) mass spectrometer. Cefotaxime-D3 was used as the internal standard, and calibration curves (0.5-200 mg/L) were established for each matrix. The method was validated in accordance with ICH M10 bioanalytical guidelines for selectivity, linearity, accuracy, precision, carry-over, matrix effect, recovery, process efficiency, stability, and VAMS-specific factors (hematocrit and drying time). The total run time was 3 min, including the internal standard, with an LLOQ of 0.5 mg/L across all matrices. The coefficients of determination (R2) were > 0.995, IS-normalized matrix factors were close to 1.0, and accuracy and precision were within ±15 % at all quality control levels. Cefazolin remained stable under all preanalytical conditions studied. VAMS values were unaffected by changes in hematocrit within clinically relevant ranges. The feasibility of the multi-matrix analytical method in routine practice has been demonstrated. The multi-matrix, microsampling-compatible LC-MS/MS assay described is sensitive and specific for TDM and PK/PD studies, aimed at defining precise dosing regimens for cefazolin in neonatal and pediatric critical care settings.

Keywords:  Cefazolin; LC-MS/MS; Perioperative antimicrobial prophylaxis; Plasma ultrafiltrate; Therapeutic drug monitoring; Unbound drugs; Volumetric absorptive microsampling

DOI:  https://doi.org/10.1016/j.cca.2025.120808
J Lipid Res. 2025 Dec 26. pii: S0022-2275(25)00231-7. [Epub ahead of print] 100968

Improved preanalytical workflow for pancreatic tissue lipidomics: Insights into lipid stability and polar lipid recovery.

Karol Parchem, Malena Manzi, Robert Jirásko, Ondřej Peterka, Zuzana Lásko, Ondřej Kuda, Michal Holčapek.

  Tissue lipidomics is a rapidly advancing field in clinical and biomedical research that provides crucial information on the lipid-driven molecular mechanisms underlying physiological and pathological conditions. However, accurate mass spectrometry-based analysis requires careful preanalytical handling due to the metabolic activity of tissue and analyte heterogeneity. Here, we introduce a robust tissue processing workflow with the pancreas as a model of a highly metabolically active organ. First, we evaluate lipid stability in porcine pancreatic tissue stored on ice, observing significant lysophospholipid formation after 60-120 minutes. Then, we compare sample handling using ice versus liquid nitrogen for both porcine and mouse pancreatic tissues, illustrating that processing temperature affects low-abundant lipid class levels, with liquid nitrogen providing better preservation. To enhance polar lipidome analysis, we optimize a hexane-methanol liquid-liquid extraction protocol and find that the addition of 2% (v/v) water to methanol yields the most effective recovery and reproducibility. Finally, the workflow is applied to mouse pancreatic tissue samples, enabling the identification of 209 polar lipid species across 10 classes, with 124 species quantified. Among these, hexosylceramides show clear sex-specific variation.

Keywords:  Lipid fractionation; Mass spectrometry; Pancreas; Sample preparation; Supercritical fluid chromatography; Tissue lipidomics

DOI:  https://doi.org/10.1016/j.jlr.2025.100968
J Sep Sci. 2025 Dec;48(12): e70340

A Protocol for Robust Discovery Proteomics Using Nano Liquid Chromatography Using Pillar-Array Column Technology and High-Resolution Mass Spectrometry With Data-Independent Acquisition.

Daniel Papp, Hanrong Wen, David Scheich, Nico Lingg, Goran Mitulović, Sebastiaan Eeltink.

  Nano liquid chromatography-mass spectrometry has come to be a key enabling technology in shotgun proteomics due to the combination of exceptional separation power, sensitivity, and comprehensiveness. However, the know-how of setting up proteomics methods to deliver robust, reliable, and meaningful results to large-scale life science experiments has remained somewhat ambiguous. This protocol outlines guidance for establishing nano-LC-MS/MS workflows focusing on comprehensive and untargeted deep proteome profiling, using state-of-the-art column technology and mass spectrometry. Employing a second-generation micropillar-array column, a trade-off is demonstrated between analysis time and chromatographic resolving power, which in turn impacts peptide and protein identification scores from a commercial HeLa reference standard. Furthermore, a straightforward workflow to develop a data-independent acquisition (DIA)-parallel accumulation-serial fragmentation (PASEF) analytical method is proposed, with a special focus on the optimization of the ESI source settings. Besides the method development, the study discusses the use of segmented gradients, and an MS-compatible surfactant in the sample diluent is also explored. Finally, the robustness of the developed method is demonstrated through consistently identifying 7558 protein groups (CV = 0.3%) as maintaining high repeatability peptide retention times (mean CV = 0.2%) and system pressure (CV = 0.4%) over 21 consecutive analyses.

Keywords:  biomarker discovery; nano liquid chromatography coupled with mass spectrometry (LC–MS); pillar‐array column; proteomics; tims‐TOF

DOI:  https://doi.org/10.1002/jssc.70340
Anal Chem. 2026 Jan 02.

spatialstein: An Open-Source Workflow for Annotation, Deconvolution, and Spatially Aware Segmentation of Mass Spectrometry Imaging Data.

Michał Aleksander Ciach, Dan Guo, Kylie Ariel Bemis, Dirk Valkenborg, Olga Vitek, Anna Gambin.

Mass Spectrometry Imaging (MSI) data sets are markedly different from optical images. However, analysis algorithms often overlook the intricacies of this kind of data. In MSI, a frequently observed phenomenon is variability in signal intensity between pixels caused by factors other than differences in analyte concentrations. Another common issue is the presence of ions with overlapping isotopic envelopes resulting in isobaric interference. The first factor causes random variations of the signal from the same anatomical regions. The second can cause the spatial distribution of a single peak to represent a mixture of spatial distributions of several analytes. Both factors affect the accuracy of data analysis methods such as MSI segmentation. In this article, we demonstrate that accounting for the intricate structure of MSI data can increase the accuracy of the analysis results. We propose an approach that leverages recent advancements in computational mass spectrometry to separate overlapping isotopic envelopes and mitigate pixel-to-pixel variability of signal intensity. We implemented the approach in spatialstein, an open-source workflow that provides a tentative annotation of an MSI data set with molecular formulas, generates a deconvolved ion image for each annotated ion, and segments each deconvolved ion image into regions of distinct intensity of the corresponding analyte. The structure of the workflow is modular, making it highly modifiable and applicable, whole or in parts, to other studies. The spatialstein workflow is available at https://github.com/mciach/spatialstein.

DOI: https://doi.org/10.1021/acs.analchem.5c04737
J Lipid Res. 2025 Dec 31. pii: S0022-2275(25)00234-2. [Epub ahead of print] 100971

Spatial Mapping of PUFA Incorporation into Phospholipids with bisallylic deuteration.

Junji Obi, Kuniyuki Kano, Yuta Shimanaka, Shukichi Haisa, Nozomu Kono, Junken Aoki.

  Mass spectrometry (MS) imaging using stable isotope-labeled fatty acids provides a groundbreaking approach to precisely localizing exogenous fatty acids and their metabolites in vivo. However, challenges persist, particularly with fatty acids labeled with fixed isotopic numbers, which can lead to spectral interferences and limit the number of metabolites that can be detected. In this study, we employed a bisallylic deuteration method to synthesize dihomo-γ-linolenic acid (DGLA) isotopes with m/z values ranging from +4 to +8 Da relative to endogenous DGLA, which allowed us to meticulously dissect DGLA metabolism in mice using LC-QTof-MS and MS imaging. Our strategy enabled the clear selection of m/z values for phospholipids enriched with deuterated DGLA (D-DGLA) and deuterated ARA (D-ARA) derived from D-DGLA, all while maintaining low background noise. This precision facilitated the successful visualization of D-DGLA and D-ARA-containing phospholipids in lung tissue, revealing their distinct localization compared to endogenous phospholipids. Our findings highlight bisallylic deuteration as a powerful tool for elucidating the in vivo dynamics of exogenous polyunsaturated fatty acids (PUFAs) through MS imaging techniques.

Keywords:  Bisallylic deuteration; Dihomo-γ-linolenic acid (DGLA); Mass spectrometry (MS) imaging; Phospholipids; Polyunsaturated fatty acids (PUFAs)

DOI:  https://doi.org/10.1016/j.jlr.2025.100971
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Dec 27. pii: S1570-0232(25)00462-3. [Epub ahead of print]1270 124908

An ID-HPLC-MS/MS based candidate reference measurement procedure for the quantification of imipenem, meropenem and ertapenem in human plasma.

Jing Lin, Qingqing Pan, Weihua Wang, Shuangshuang Chen, Guangliang Wu, Min Shen, Huoyan Ji.

   BACKGROUND: Carbapenems require precise plasma levels for optimal therapy, yet no LC-MS/MS method offered SI-traceability or reference-grade performance. In this study, a candidate reference measurement procedure (RMP) using isotope dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) was developed and validated for accurate quantification of imipenem (IPM), meropenem (MEM) and ertapenem (ETP) in human plasma.
METHODS: To ensure traceability to SI units, the absolute content of the IPM, MEM and ETP primary reference materials (PRMs) were determined by quantitative nuclear magnetic resonance (qNMR) spectroscopy. Samples were prepared by protein precipitation followed by dilution. Method validation was performed according to guidances from the Clinical and Laboratory Standards Institute (CLSI), including selectivity/specificity, matrix effect, carryover, linearity, limit of quantification (LOQ) and limit of detection (LOD), trueness and precision, and stability. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). Moreover, the candidate RMP was compared between two independent laboratories.
RESULTS: The method showed high selectivity and stability. Linear ranges were 0.208 to 52.087 μg/mL (IPM, r = 0.9997), 0.412 to 103.427 μg/mL (MEM, r = 0.9999) and 0.083 to 20.791 μg/mL (ETP, r = 0.9999). Mean recoveries were 100.05 to 104.80 %, 92.34 to 94.77 % and 95.55 to 100.04 %, respectively. The method demonstrated high sensitivity, with limits of LOQ of 0.103, 0.103 and 0.021 μg/mL and corresponding LOD of 0.0077, 0.0018 and 0.0006 μg/mL for IPM, MEM and ETP, respectively. No obvious matrix effect and carryover were observed. The expanded uncertainties (k = 2) were ≤ 6.12 %.
CONCLUSION: The qNMR-based candidate RMP provides SI-traceable, accurate simultaneous quantification of IPM, MEM and ETP, enabling standardization of clinical assays and reliable patient-sample measurement.

Keywords:  Candidate reference measurement procedure; Carbapenems; ID-HPLC-MS/MS; Traceability; qNMR

DOI:  https://doi.org/10.1016/j.jchromb.2025.124908
Anal Chem. 2026 Jan 02.

Coupling High-Throughput Magnetic-Bead-Based Immunoaffinity with LC-MS/MS Analysis for Quantifying Cylindrospermopsin in Different Fish Tissues.

Jinping Liu, Shengyang Liu, Sainan Zhang, Liang Peng, Lamei Lei.

Cylindrospermopsin (CYN) can bioaccumulate in aquatic food chains and cause potential toxicity to human and animal health. When analyzing biological samples using liquid chromatography with tandem mass spectrometry (LC-MS/MS), the presence of complex matrices is a major concern. In this study, we developed a novel immunoaffinity extraction method for CYN enrichment in different biological matrices. Magnetic beads bound with a monoclonal antibody against CYN were used as affinity probes to specifically enrich CYN in samples, which effectively eliminated matrix effects for LC-MS/MS analysis. Under optimized conditions, the automated method processes 32 samples in 54 min without any centrifugation or filtering steps. When coupled to LC-MS/MS, this new method shows excellent sensitivity (LOQ, 0.025 μg L-1), linearity (R2 > 0.99, 0.125-100 μg L-1), precision (RSD < 6%), and recoveries (85-105%). Using the established method to measure CYN contents in 10 muscle and liver tissue samples of fish from a reservoir of southern China, we found its analogue deoxy-cylindrospermopsin (deoxy-CYN) was detected more frequently and also with higher concentration than CYN. An average concentration of 2.38 μg kg-1 in muscle and 71.99 μg kg-1 in liver suggested that CYN accumulation is highly tissue-specific. This is the first report of immunoaffinity extraction for CYN enrichment. Our results demonstrate that the developed method is reliable and offers significant application prospects.

DOI: https://doi.org/10.1021/acs.analchem.5c06095
Anal Chem. 2025 Dec 28.

Filtration-Based Rapid ECM-Hydrogel Elimination Enables Robust MALDI MSI of Intestinal Organoids.

Fangyuan Zhang, Xinxin Shen, Jie Yuan, Jiayi Ye, Jian Sui, Huimin Tao, Jianfeng Xu, Yang Ye, Youhong Hu, Bo Wang, Jingqing Hu, Jia Liu.

The rapid development of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) offers a powerful approach for the 2D metabolic profiling of intestinal organoids. However, its application remains constrained by challenges in sample preparation. Conventional extracellular matrix (ECM)-based hydrogel removal methods, such as repeated centrifugation or enzymatic digestion, are labor-intensive and time-consuming, often causing structural disruption of organoids and degradation of metabolites, thereby compromising the spectral quality and metabolite coverage. In this study, we established a filtration-based rapid ECM-hydrogel elimination (FREE) method using a 70 μm filter, which reduced treatment time from 75 to 25 min while preserving organoid structural integrity, cell viability, and metabolic stability and, more importantly, improved data quality, with a 26.6% increase in metabolite coverage and enhanced signal intensity. Applying this workflow to an LPS-induced inflammatory intestinal organoid model, we observed spatial metabolic remodeling associated with epithelial polarity disruption. Notably, phosphatidylserine (PS) became enriched at the apical membrane, while phosphatidylethanolamine (PE) exhibited a basolateral side, suggesting a link between loss of polarity and early apoptotic signaling. Overall, FREE provides an efficient pretreatment strategy that enables MSI research on intestinal organoids to dissect the mechanisms of inflammation-associated metabolic remodeling.

DOI: https://doi.org/10.1021/acs.analchem.5c05415
Metabolomics. 2025 Dec 29. 22(1): 15

Urinary metabolome dynamics in 13C-labeled mice.

Annelaure Damont, Anaïs Legrand, Kathleen Rousseau, Laurent Bellanger, Séverine Boiry, Frédéric Gibiat, Jean-Jacques Leguay, Christophe Junot, François Fenaille, Eric Ezan.

   INTRODUCTION: Analysis of specific pathways of metabolic flux is usually achieved by administering stable isotope-labeled precursors and measuring their incorporation into selected metabolites by high-resolution mass spectrometry coupled to liquid chromatography (LC-HRMS).
OBJECTIVES: In this study, we undertook a novel approach aiming at covering the whole metabolome dynamics by providing mice with a diet fully enriched in carbon-13 (13C).
METHODS: Three animals were fed for six weeks with small pellets composed of a mixture of 13C-spirulina and 13C-wheat. All animals grew normally and their urine was collected daily. Three control mice were treated in the same way, but with an unenriched diet. LC-HRMS-based metabolomics profiling were conducted on all collected samples as well as isotope tracing.
RESULTS: Comparative LC-HRMS analysis of the 12C- and 13C-samples unambiguously identified 238 metabolites, whose 13C-labeling profiles were then studied over a 39-day period. Although overall urine labeling was fast and rapidly reached a high level of 13C-content (> 90% after 22 days), isotopic monitoring of each molecular species demonstrated that 13C-incorporation kinetics are considerably variable between metabolites, reflecting their biological function, origin and rate of biosynthesis in vivo.
CONCLUSION: This study demonstrates that mice tolerate a diet entirely labeled with carbon-13 (> 97 atom % 13C) over a period of six-weeks. It reveals the dynamics of a large part of the metabolome at the mammalian species level through urine analysis coupled to in vivo 13C-labeling. The study provides valuable insights by comprehensively covering metabolic pathways, and stands out from targeted fluxomic studies which are carried out by administering a very limited number of specific labeled precursors.

Keywords:   13C-metabolomics; In vivo; Isotope tracing; Mammal labeling; Mass spectrometry

DOI:  https://doi.org/10.1007/s11306-025-02391-4
Int J Mol Sci. 2025 Dec 16. pii: 12114. [Epub ahead of print]26(24):

Mass Spectrometry Imaging Elucidates the Precise Localization and Site-Specific Functions of Skin Lipids.

Shown Tokoro, Tadayuki Ogawa, Shujiro Hayashi, Ken Igawa.

  Lipids are essential for the skin, playing a crucial role in forming plasma membranes and maintaining the skin's permeability barrier and hydration. Intercellular lipids fill the spaces between corneocytes and contribute to the barrier function. Lipid abnormalities in the skin have been observed in many skin diseases, including atopic dermatitis and psoriasis. However, the specific localization and roles of skin lipids at particular sites remain incompletely elucidated due to the limited methods available for comprehensive lipid analysis. This study aims to precisely determine the localization of skin lipids, especially intercellular lipids, and investigate their roles and metabolism using mass spectrometry imaging (MSI). We conducted high-resolution (spatial resolution: 5 µm) matrix-assisted laser desorption/ionization (MALDI)-MSI on the lower back and buttocks and created overlay images of skin lipids to clarify their precise localizations. Ceramide was localized in the outermost layer among intercellular lipids. Cholesterol and free fatty acids were present in the stratum corneum but were at trace levels in the outermost layer. Cholesterol sulfate was abundant in the granular layer and gradually decreased in the stratum corneum, promoting desquamation. Phospholipids were confined to the viable epidermis (stratum corneum-/epidermis+), which forms the plasma membrane. A significant increase in mass intensity in the stratum corneum was observed for ceramide, sphingoid base, cholesterol, and free fatty acids, along with a decrease in phospholipids compared with those in the viable epidermis, based on region of interest analysis (Mann-Whitney test, p < 0.0005). We clarified the precise localization of skin lipids, particularly intercellular lipids. Our findings supported the reported functions of skin lipids at specific sites. Skin lipids are metabolized to form intercellular lipids in the stratum corneum, which are essential for the skin barrier. Our current lipid localization data serve as a baseline, or healthy control dataset, for future MSI-based lipid biomarker research in disease groups.

Keywords:  ceramide; cholesterol; cholesterol sulfate; free fatty acid; intercellular lipid; mass spectrometry imaging; phospholipid; skin lipid

DOI:  https://doi.org/10.3390/ijms262412114
Rapid Commun Mass Spectrom. 2026 Mar 30. 40(6): e70023

Determination of 15 Restricted Substances in Plastic-Packaged Food by Gas Chromatography-Mass Spectrometry.

Xin Yang, Hanning Zhang, Jinqiang Yan, Hongmei Xue, Hongmei Shi, Junjie Miao.

   RATIONALE: Chemicals migrating from plastic and take-out food packaging can leach into food, posing inherent health risks to consumers. Given the ubiquitous use of plastic packaging in daily diets, reliable analytical methods for detecting restricted substances in food contact materials are imperative for safeguarding public health.
METHODS: A novel analytical method, coupling gas chromatography-mass spectrometry (GC-MS) with liquid-liquid extraction for sample pretreatment, was established for the simultaneous separation and quantification of 15 common restricted substances (10 plasticizers, 1 antioxidant, and 4 ultraviolet absorbers) migrating from food plastic packaging materials into food products.
RESULTS: The method exhibited excellent linearity (correlation coefficients, r > 0.99) across wide concentration ranges, with limits of detection (LODs) ranging from 0.005 to 0.046 mg/L. Validation data showed average recoveries of 72.30%-109.03% and relative standard deviations (RSDs, n = 6) of 1.13%-26.50%, confirming its analytical reliability.
CONCLUSIONS: This method enables rapid and accurate determination of the 15 target restricted substances, facilitating migration monitoring in food plastic packaging and strengthening technical support for food safety assessment in daily and take-out food contexts.

Keywords:  determination; food plastic packaging; gas chromatography–mass spectrometry; migration; restricted substances

DOI:  https://doi.org/10.1002/rcm.70023
Anal Chem. 2025 Dec 31.

Temperature-Responsive Liquid Chromatography for One- and Two-Dimensional Separations in Compound-Specific Isotope Analysis.

Sarah P Rockel, Adriaan Ampe, Matthias Stüwe, Thomas Piper, Maik A Jochmann, Frédéric Lynen, Torsten C Schmidt.

Temperature-responsive liquid chromatography (TRLC) enables the possibility of retention modulation under fully aqueous isocratic conditions through incorporation of smart polymers such as poly(N-isopropylacrylamide) (PNIPAAm) into the stationary phase. This feature seems to be particularly advantageous for liquid chromatography isotope ratio mass spectrometry (LC-IRMS), which is constrained by its requirement for fully aqueous eluents and limited stationary phase options. In this study, we demonstrate for the first time the coupling of TRLC with LC-IRMS for compound-specific carbon isotope analysis. Using testosterone and related steroids as a model substance class, we show that PNIPAAm-based TRLC columns provide stable δ13C measurements across isothermal and temperature gradient programs without introducing isotopic bias (σ ≤ 0.5‰ across all measurements). One-dimensional TRLC-IRMS enabled separation of several steroid standards under fully aqueous conditions and the determination of testosterone in an ethanol-based gel. A heart-cut two-dimensional (2D) LC-IRMS configuration, combining reversed phase LC in the first dimension with TRLC in the second dimension, achieves baseline separation of otherwise coeluting steroids and accurate δ13C determination in complex, lipid-rich pharmaceutical matrices. This work establishes TRLC as a powerful addition to the LC-IRMS toolkit, expanding method flexibility and selectivity without sacrificing isotopic accuracy and opening new avenues for compound specific isotope analysis via LC-IRMS.

DOI: https://doi.org/10.1021/acs.analchem.5c05840
Brief Bioinform. 2025 Nov 01. pii: bbaf682. [Epub ahead of print]26(6):

MetImputBERT: a pretrained BERT framework for missing value imputation in NMR metabolomics data.

Alzheimer's Disease Neuroimaging Initiative

  Missing values in nuclear magnetic resonance metabolomics data compromise downstream clinical interpretation. Here, we present MetImputBERT, an imputation method based on a pretrained BERT framework. MetImputBERT uses the masks in the masked language model to simulate missing values and leverages predictions and reconstructions to these positions to simulate the imputation process. The learning of MetImputBERT is driven by minimizing the reconstruction error. MetImputBERT was pretrained on the largest metabolomics dataset to date, comprising data from over 230 000 individuals in the UK Biobank. When new datasets with missing values were encountered, MetImputBERT loaded the pretrained parameters and directly imputed the missing values by inferring their reconstructed estimates. MetImputBERT outperformed commonly used methods-K-nearest neighbors, multiple imputation by chained equations, and singular value decomposition-in imputation performance on two independent test sets. We provide an open-source Python tool that allows users to quickly impute missing values in their own NMR metabolomics data without any additional training.

Keywords:  BERT; imputation; metabolomics; missing values

DOI:  https://doi.org/10.1093/bib/bbaf682
Biomed Chromatogr. 2026 Feb;40(2): e70344

Development and Validation of an Assay Method for Levothyroxine Soft Gelatin Capsules Utilizing the RP-HPLC Technique.

Nagaraju Katari, J Subba Rao, S Vidyadhara, Dileep J Babu Bikkina.

  A robust, precise, accurate, and stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative estimation of levothyroxine sodium in soft gelatin capsule dosage forms across a wide strength range (13 to 200 μg). The objective of this study was to establish a single, specific method capable of assessing both assay with high sensitivity, particularly at a low-test concentration of 1 ppm, while ensuring specificity toward all known impurities listed in the levothyroxine drug substance monograph. Chromatographic separation was achieved using an InertSustain AQ C18 column (250 × 4.6 mm, 5 μm) maintained at 60°C. The mobile phase system comprised 90% phosphate buffer and 10% acetonitrile (Mobile Phase A) and 90% acetonitrile with 10% water (Mobile Phase B), delivered at a flow rate of 1.2 mL/min. Detection was carried out at 225 nm with a total runtime of 25 min. The retention time of levothyroxine was approximately 7.1 min. The method was validated across a linearity range of 0.5 to 2.0 ppm (50% to 200% of the test concentration), yielding a correlation coefficient (r2) > 0.999. The method demonstrated excellent precision, with %RSD values of 0.2% for standard solutions and 0.8% for six sample preparations. Limits of detection (LOD) and quantification (LOQ) were determined to be 0.05 and 0.1 ppm, respectively. The method was found to be specific to all 14 known impurities, robust, accurate, and stability-indicating. This validated method is suitable for routine quality control analysis and regulatory submissions, meeting industrial standards for accuracy, precision, and specificity across all marketed strengths of levothyroxine sodium capsules.

Keywords:  RP‐HPLC; levothyroxine sodium; method validation and Impurities; soft gelatin capsule; stability‐indicating

DOI:  https://doi.org/10.1002/bmc.70344
J Am Soc Mass Spectrom. 2025 Dec 29.

An Interlaboratory Comparison of High-Resolution Ion Mobility Collision Cross-Sections from Human Plasma.

Rachel A Harris, Emanuel Zlibut, Allison R Reardon, Kyle E Lira, Jody C May, Sarah M Stow, David L Williamson, Jennifer Krone, Komal Kedia, John A McLean, Frederick G Strathmann.

The increasing adoption of high-resolution ion mobility (HRIM) in untargeted omics workflows underscores the need for precise collision cross-section (CCS) measurements which are highly reproducible across various laboratories and instrumentation. To evaluate the reproducibility of a high-resolution ion mobility platform, an interlaboratory study was undertaken using structures for lossless ion manipulation-based traveling wave ion mobility spectrometry (TWSLIM) in nitrogen drift gas. Across 250 lipid features spanning glycerophospholipids, glycerolipids, and sphingolipids detected from a lipidomic extract of human plasma standard reference material, the platform demonstrated high CCS measurement reproducibility, with an average relative standard deviation (%RSD) of ∼0.1%. Triglycerides in general were found to exhibit multiple IM features that served as an illustrative example where analysis via HRIM and data-independent, mobility-aligned fragmentation (MAF) provides critical insights into their chemical structures. To support the future development of HRIM in lipidomic workflows, a large (n = 250) number of lipid consensus TWSLIMCCSN2 values was compiled from the interlaboratory study into a HRIM database for community use.

DOI: https://doi.org/10.1021/jasms.5c00396
J Mass Spectrom. 2026 Jan;61(1): e70004

Desorption Electrospray Ionization Mass Spectrometry Imaging: Principles, Advancements, and Multidisciplinary Applications.

Yan Wang, Huannan Wang, Shuncun Zhang, Yidan Xi, Zhen Zhang.

  Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI), as an ambient ionization technique, enables label-free, matrix-free, and minimal sample pretreatment molecular mapping of biological surfaces under atmospheric pressure. Since its inception in 2004, DESI-MSI has evolved through innovations in ionization sources, sprayer design, and data processing, allowing high-throughput visualization of spatial distributions for diverse molecules (e.g., lipids, metabolites, drugs) in tissues. This review systematically reviews the fundamental principles, instrumental configurations, and critical technical parameters of DESI-MSI. Recent technological advancements, specifically the development of nanoscale DESI achieving spatial resolutions under 50 μm and the implementation of additive-enhanced solvents such as metal ions and reactive anions to enhance sensitivity and specificity, are critically evaluated. Furthermore, the multidisciplinary applications of DESI-MSI are comprehensively examined: (1) in biomedicine, it facilitates cancer margin delineation (e.g., breast, prostate tumors) and neuropathological biomarker discovery; (2) in pharmaceutical research, it enables in situ drug distribution analysis and tissue metabolism profiling; (3) in plant sciences, it supports spatial mapping of phytochemicals, pesticide residues, and host-microbe interactions. Nevertheless, despite its advantages in real-time analysis and nondestructive sampling, inherent challenges persist, including limited spatial resolution (~200 μm) and complexities in interpreting complex datasets. Future work should emphasize the integration of multimodal imaging approaches, machine learning-driven data processing pipelines, and clinical translation strategies for intraoperative diagnostics. Collectively, this study positions DESI-MSI as a transformative spatial metabolomics tool with expanding potential in clinical diagnostics and high-throughput drug screening.

Keywords:  ambient ionization; desorption electrospray ionization mass spectrometry imaging; molecular imaging; spatial distributions

DOI:  https://doi.org/10.1002/jms.70004