bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–03–02
fifty-four papers selected by
Sofia Costa, Matterworks
  1. Chiral supercritical fluid chromatography-mass spectrometry with liquid chromatography fractionation for the characterization of enantiomeric composition of fatty acid esters of hydroxy fatty acids.
  2. A New, Validated GC-PICI-MS Method for the Quantification of 32 Lipid Fatty Acids via Base-Catalyzed Transmethylation and the Isotope-Coded Derivatization of Internal Standards.
  3. Detection of Etomidate and Etomidate Acid in Urine Using HPLC-MS/MS Method.
  4. Deep Learning-Based Molecular Fingerprint Prediction for Metabolite Annotation.
  5. Quantification of three neurotransmitters in cerebrospinal fluid, serum and random urine using a robust and simplified liquid chromatography-tandem mass spectrometry method.
  6. Multi-Pass Arrival Time Correction in Cyclic Ion Mobility Mass Spectrometry for Imaging and Shotgun Lipidomics.
  7. Machine Learning in Small-Molecule Mass Spectrometry.
  8. Human Untargeted Metabolomics in High-Throughput Gut Microbiome Research: Ethanol vs Methanol.
  9. Quantification of Glycosaminoglycans in Urine by Isotope Dilution Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry.
  10. Integrating the lactulose-mannitol test for intestinal permeability with untargeted metabolomics for drug monitoring through dual liquid chromatography-mass spectrometry.
  11. Improving the detectability of low-abundance p-cresol in biological matrices by chemical derivatization and LC-MS/MS determination.
  12. Determination of 14 Benzodiazepine Multiresidues in Aquaculture Environment by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
  13. Validation of an LC-HRMS Method for Quantifying Indoxyl Sulfate and p-Cresyl Sulfate in Human Serum.
  14. Simultaneous determination of 25(OH)D2, 25(OH)D3 and 1α,25(OH)2D3 in human serum by derivatization-liquid chromatography-tandem mass spectrometry.
  15. Challenges in PFAS Postdegradation Analysis: Insights from the PFAS-CTAB Model System.
  16. Comprehensive analysis of oxidized arachidonoyl-containing glycerophosphocholines using ion mobility spectrometry-mass spectrometry.
  17. 1H-NMR Lipidomics, Comparing Fatty Acids and Lipids in Cow, Goat, Almond, Cashew, Soy, and Coconut Milk Using NMR and Mass Spectrometry.
  18. Structural Annotation Method for Locating sn- and C═C Positions of Lipids Using Liquid Chromatography-Electron Impact Excitation of Ions from Organics (EIEIO)-Mass Spectrometry.
  19. Nanoconfined in-situ sampling and extracting switching needle device construction for highly enrichment of polar metabolites in human urine.
  20. Matrix Linear Models for Connecting Metabolite Composition to Individual Characteristics.
  21. Enhancing the Accuracy of Identification and Relative Quantification of Unsaturated Fatty Acids in Serum via a Stable Isotope-Labeled Double Derivatization Strategy.
  22. UPLC-MS/MS method for simultaneous determination of pyrantel, praziquantel, febantel, fenbendazole and oxfendazole in dog plasma and its application to a bioequivalence study.
  23. Evaluating sample normalization methods for MS-based multi-omics and the application to a neurodegenerative mouse model.
  24. Microflow Liquid Chromatography Coupled to Multinozzle Electrospray Ionization for Improved Lipidomics Coverage of 3D Clear Cell Renal Cell Carcinoma.
  25. A Systematic Study of Liquid Chromatography in Search of the Best Separation of Cannabinoids for Potency Testing of Hemp-Based Products.
  26. Simultaneous Determination of Six Acidic Herbicides and Metabolites in Plant Origin Matrices by QuEChERS-UPLC-MS/MS.
  27. Pharmacokinetic Study of HRO761 in Rats by Liquid Chromatography Combined With Electrospray Ionization Tandem Mass Spectrometry.
  28. Rapid single-step pretreatment of urine samples using a 2D carbon microfiber fractionation for phthalate and metabolite detection via GC-MS and LC-MS/MS.
  29. Reference Intervals of Serum Metabolites and Lipids of a Healthy Chinese Population Determined by Liquid Chromatography-Mass Spectrometry.
  30. Targeted Quantitative Analysis of Urinary Bile Acids by Liquid Chromatography-Tandem Mass Spectrometry: Method Development and Application to Healthy and Obese Children.
  31. Visualization of Efavirenz-Induced Lipid Alterations in the Mouse Brain Using MALDI Mass Spectrometry Imaging.
  32. Urinary Metabolomic Profile is Minimally Impacted by Common Storage Conditions and Additives.
  33. A Novel LC-MS/MS Method for the Measurement of Elexacaftor, Tezacaftor and Ivacaftor in Plasma, Dried Plasma Spot (DPS) and Whole Blood in Volumetric Absorptive Microsampling (VAMS) Devices.
  34. Exploring Single-Probe Single-Cell Mass Spectrometry: Current Trends and Future Directions.
  35. New Standardized Procedure to Extract Glyphosate and Aminomethylphosphonic Acid from Different Matrices: A Kit for HPLC-UV Detection.
  36. IodoFinder: Machine Learning-Guided Recognition of Iodinated Chemicals in Nontargeted LC-MS/MS Analysis.
  37. Ultrasensitive Determination of Amino Acids in Single Cells by Chemical Isotope Labeling with Liquid Chromatography Mass Spectrometry Analysis.
  38. A Systematic Approach to Discover New Natural Product Scaffolds Using Database-Derived Relative Mass Spectral Defects and Molecular Networking.
  39. Two-dimensional isotopic shifts for steroid isomer delineation with high-resolution cyclic ion mobility separations.
  40. Utilizing High-Resolution Mass Spectrometry Data Mining Strategy in R Programming Language for Rapid Annotation of Absorbed Prototypes and Metabolites of Gypenosides.
  41. Molecular networking: An efficient tool for discovering and identifying natural products.
  42. GlycoDash: automated, visually assisted curation of glycoproteomics datasets for large sample numbers.
  43. pyOpenMS-viz: Streamlining Mass Spectrometry Data Visualization with pandas.
  44. Ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) for simultaneous estimation of residual glyphosate and its metabolite (amino methyl phosphonic acid - AMPA) in various vaccines.
  45. Analysis of germination and vigor of cashew seeds for germplasm conservation by NMR and uPLC-qTOF-MS/MS based metabolomics.
  46. Optimized Extraction Method for Neutral Cannabinoids Quantification Using UHPLC-HRMS/MS.
  47. To Impute or Not To Impute in Untargeted Metabolomics─That is the Compositional Question.
  48. Network Flow Methods for NMR-Based Compound Identification.
  49. Imatinib and norimatinib therapeutic monitoring using dried blood spots: Analytical and clinical validation, and performance comparison of volumetric collection devices.
  50. Microbiota-Gut-Brain Axis: Mass-Spectrometry-Based Metabolomics in the Study of Microbiome Mediators-Stress Relationship.
  51. Contamination Status and Health Risk Assessment of 73 Mycotoxins in Four Edible and Medicinal Plants Using an Optimized QuEChERS Pretreatment Coupled with LC-MS/MS.
  52. Analytical validation and pilot clinical application of a UPLC-MS/MS method for determining intracellular mycophenolic acid and metabolites in kidney transplant recipients.
  53. Development of a novel UHPLC-MS/MS method for quantitative analysis of pirtobrutinib in rat plasma: application to pharmacokinetic study.
  54. [Ruhemann's Purple Monitoring by UHPLC/MS/MS for Ninhydrin Test].
  1. Anal Chim Acta. 2025 Apr 01. pii: S0003-2670(25)00129-1. [Epub ahead of print]1345 343735
    Chiral supercritical fluid chromatography-mass spectrometry with liquid chromatography fractionation for the characterization of enantiomeric composition of fatty acid esters of hydroxy fatty acids.
    Oleksandr Kozlov, Miroslav Lísa, Martin Riecan, Ondrej Kuda.
       BACKGROUND: Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with promising therapeutic potential for diabetes and inflammation. They represent complex mixtures of different isomers whose biological functions are the subject of investigation. Highly selective methods are required to characterize the composition of enantiomers in biological samples composed of many isobars and regioisomers. We aimed to develop a method for characterizing the enantiomeric composition of FAHFAs in biological samples using supercritical fluid chromatography-mass spectrometry (SFC-MS).
    RESULTS: The influence of key chromatographic parameters, such as column chemistry, mobile phase composition, and gradient, on the separation efficiency of 21 commercially available FAHFA regioisomers without stated absolute configuration and 4 FAHFA enantiomers was assessed. The optimized SFC-MS method utilizes a chiral column based on a tris-(3-chloro-5-methylphenylcarbamate) derivative of amylose (Lux i-Amylose-3) and acetonitrile-methanol mobile phase modifier, enabling fast enantioseparation of most FAHFA racemic pairs in 5 min. However, the SFC separation of FAHFA regioisomers was less effective, limiting its applicability to complex biological samples. To address this, we propose an offline two-dimensional separation approach with reversed-phase liquid chromatography for isolating FAHFA regioisomers, followed by chiral SFC-MS analysis of fractions. The suitability of the method was demonstrated by characterizing the enantiomeric composition of FAHFA in white adipose tissue and rice samples. The chiral analysis revealed the presence of both R- and S-FAHFA isomers in the samples, with one enantiomer being predominant.
    SIGNIFICANCE: The developed approach represents a proof of concept for the use of SFC-MS with LC prefractionation for the characterization of FAHFA enantiomeric composition in complex biological samples, providing a valuable tool for future research on the biological roles of bioactive lipids in health and disease.
    Keywords:  Chiral separation; FAHFA; Isomers; Lipids; Offline two-dimensional chromatography; Supercritical fluid chromatography
    DOI:  https://doi.org/10.1016/j.aca.2025.343735
  2. Metabolites. 2025 Feb 07. pii: 104. [Epub ahead of print]15(2):
    A New, Validated GC-PICI-MS Method for the Quantification of 32 Lipid Fatty Acids via Base-Catalyzed Transmethylation and the Isotope-Coded Derivatization of Internal Standards.
    Petr Vodrážka, Lucie Řimnáčová, Petra Berková, Jan Vojtíšek, Miroslav Verner, Martin Moos, Petr Šimek.
      Background: Fatty acids (FAs) represent a ubiquitous class of nonpolar alkyl carboxylate metabolites with diverse biological functions. Nutrition, metabolism, and endogenous and exogenous stress influence the overall FA metabolic status and transport via the bloodstream. FAs esterified in lipids are of particular interest, as they represent promising biomarkers of pathological diseases and nutritional status. Methods: Here, we report a validated gas chromatographic-mass spectrometric (GC-MS) method for the quantitative analysis of 32 FAs exclusively bound in esterified lipids. The developed sample preparation protocol comprises three steps using only 5 µL of human serum for Folch extraction, sodium methoxide-catalyzed transesterification in tert-butyl methyl ether, and re-extraction in isooctane prior to a quantitative GC-MS analysis with positive ion chemical ionization (PICI) and selected ion monitoring (SIM). Results: The base-catalyzed transmethylation step was studied for 14 lipid classes and was found to be efficient under mild conditions for all major esterified lipids but not for free FAs, lipid amides, or sphingolipids. To minimize matrix effects and instrument bias, internal fatty acid trideuteromethyl esters (D3-FAME) standards were prepared through isotope-coded derivatization with D3-labeled methylchloroformate/methanol medium mixed with each transmethylated serum extract for the assay. The method was validated according to FDA guidelines and evaluated by analyzing NIST SRM 2378 Serum 1 and sera from three healthy donors. Conclusions: The measured quantitative FA values are consistent with the reference data of SRM 2378, and they demonstrate the application potential of the described method for general FA analysis in esterified lipids as a novel complementary tool for lipidomics, as well as for the analysis of membrane FAs in dry blood spots and red blood cells.
    Keywords:  GC-MS; NIST SRM 2378; fatty acid analysis; human serum; isotope-coded derivatization; positive ion chemical ionization; quantitative analysis; transmethylation
    DOI:  https://doi.org/10.3390/metabo15020104
  3. Fa Yi Xue Za Zhi. 2024 Oct 25. pii: 1004-5619(2024)05-0454-07. [Epub ahead of print]40(5): 454-460
    Detection of Etomidate and Etomidate Acid in Urine Using HPLC-MS/MS Method.
    Tian-Fu He, Huan-Hui Zhu, Yuan-Yuan Tian, Yin-Shuang Jin, Xian-Wen Lin, Song-Cai Wang.
       OBJECTIVES: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the detection of etomidate and etomidate acid in urine samples.
    METHODS: Protein in the urine samples was precipitated by adding acetonitrile, and the supernatant was obtained after centrifugation and filtered. The supernatant was separated on a C18 column with a mobile phase consisting of 0.1% formic acid solution and acetonitrile at a flow rate of 0.4 mL/min. The detection was performed in positive electrospray ionization (ESI) and multiple reaction monitoring (MRM) modes. The method was validated for selectivity, linearity and limit of detection (LOD), and applied to a case of etomidate poisoning death.
    RESULTS: The LOD of etomidate and etomidate acid were 0.2 and 0.5 ng/mL, respectively, and the limit of quantitation (LOQ) were 0.5 and 1.0 ng/mL, respectively. Good linear relationship was observed within the linear range (r>0.995 0). At three concentration levels (0.5, 5, 50 ng/mL for etomidate and 1, 10, 100 ng/mL for etomidate acid), the matrix effect was within the range of 5.42% to 18.47%, the extraction recovery rate was greater than 84.25% and the stability was greater than 88.23%. The accuracy, precision and dilution reliability all met the experimental requirements. Etomidate and etomidate acid were successfully detected with the concentrations of 8.82 and 27.88 μg/mL in the urine of a deceased individual who had consumed excessive etomidate.
    CONCLUSIONS: The method has simple pretreatment, high sensitivity and wide linear range, which can be applied to the detection of etomidate and etomidate acid in urine samples in forensic science.
    Keywords:  etomidate; etomidate acid; forensic medicine; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); toxicological analysis; urine
    DOI:  https://doi.org/10.12116/j.issn.1004-5619.2023.330702
  4. Metabolites. 2025 Feb 14. pii: 132. [Epub ahead of print]15(2):
    Deep Learning-Based Molecular Fingerprint Prediction for Metabolite Annotation.
    Hoi Yan Katharine Chau, Xinran Zhang, Habtom W Ressom.
      Background/Objectives: Liquid chromatography coupled with mass spectrometry (LC-MS) is a commonly used platform for many metabolomics studies. However, metabolite annotation has been a major bottleneck in these studies in part due to the limited publicly available spectral libraries, which consist of tandem mass spectrometry (MS/MS) data acquired from just a fraction of known compounds. Application of deep learning methods is increasingly reported as an alternative to spectral matching due to their ability to map complex relationships between molecular fingerprints and mass spectrometric measurements. The objectives of this study are to investigate deep learning methods for molecular fingerprint based on MS/MS spectra and to rank putative metabolite IDs according to similarity of their known and predicted molecular fingerprints. Methods: We trained three types of deep learning methods to model the relationships between molecular fingerprints and MS/MS spectra. Prior to training, various data processing steps, including scaling, binning, and filtering, were performed on MS/MS spectra obtained from National Institute of Standards and Technology (NIST), MassBank of North America (MoNA), and Human Metabolome Database (HMDB). Furthermore, selection of the most relevant m/z bins and molecular fingerprints was conducted. The trained deep learning models were evaluated on ranking putative metabolite IDs obtained from a compound database for the challenges in Critical Assessment of Small Molecule Identification (CASMI) 2016, CASMI 2017, and CASMI 2022 benchmark datasets. Results: Feature selection methods effectively reduced redundant molecular and spectral features prior to model training. Deep learning methods trained with the truncated features have shown comparable performances against CSI:FingerID on ranking putative metabolite IDs. Conclusion: The results demonstrate a promising potential of deep learning methods for metabolite annotation.
    Keywords:  LC-MS/MS; deep learning; metabolite identification; molecular fingerprint prediction
    DOI:  https://doi.org/10.3390/metabo15020132
  5. J Chromatogr A. 2025 Feb 18. pii: S0021-9673(25)00139-6. [Epub ahead of print]1747 465791
    Quantification of three neurotransmitters in cerebrospinal fluid, serum and random urine using a robust and simplified liquid chromatography-tandem mass spectrometry method.
    Jian Zhong, Lihua Guan, Yutong Zou, Jialei Yu, Xiaoli Ma, Ruichen Liu, Songlin Yu, Ling Qiu.
      Imbalance in the monoamine neurotransmitters has been implicated in a variety of neurological disorders, making it necessary to quantify neurotransmitters accurately. Thus, the study aims to develop a robust and validated method for simultaneously quantifying serotonin, 5-Hydroxyindole Acetic Acid (5-HIAA), and homovanillic acid (HVA) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). One-step precipitation with methanol containing 0.2 % formic acid was used for pretreating serum and cerebrospinal fluid samples, while dilution with deionized water was used for pretreating urine samples. Neurotransmitters were detected using an Exion AD liquid chromatography-tandem Qtrap 6500 Plus mass spectrometer, with a total run time of 6.5 mins. The linearity range was 0.5-500.0, 0.2-100.0, 2.0-1000.0 ng/mL for serotonin, 5-HIAA, and HVA in serum and cerebrospinal fluid, and 2.0-500.0, 40.0-10,000.0, 100.0-10,000.0 ng/mL in urine (R2≥0.997). Recovery rate was 81.5-114.4 %, 80.3-114.6 %, and 85.0-115.6 % for serotonin, 5-HIAA, and HVA in three matrices. The matrix effect was compensated by using internal standards. Acceptable intra-assay and inter-assay precision were achieved for all analytes and the total coefficients of variation were 4.9-14.4 %, 6.1-11.2 %, and 4.5-10.5 % for serotonin, 5-HIAA, and HVA. Additionally, we also explored the distribution of neurotransmitters. Patients with motor impairment had higher HVA levels than those without symptoms (P < 0.05), while serotonin and 5-HIAA concentrations were insignificant. Accordingly, a robust LC-MS/MS method combined with easy sample preprocessing was established and systematically validated for quantifying three important neurotransmitters in multiple matrices in a single run, allowing for accurate identification of motor impairment.
    Keywords:  5-hydroxyindole acetic acid; Homovanillic acid; LC-MS/MS; Serotonin
    DOI:  https://doi.org/10.1016/j.chroma.2025.465791
  6. ACS Meas Sci Au. 2025 Feb 19. 5(1): 109-119
    Multi-Pass Arrival Time Correction in Cyclic Ion Mobility Mass Spectrometry for Imaging and Shotgun Lipidomics.
    Pattipong Wisanpitayakorn, Narumol Jariyasopit, Kassaporn Duangkumpha, Jun Xian Goh, Martin E Palmer, Yongyut Sirivatanauksorn, Sakda Khoomrung.
      Direct-infusion mass spectrometry (DI-MS) and mass spectrometry imaging (MSI) are powerful techniques for lipidomics research. However, annotating isomeric and isobaric lipids with these methods is challenging due to the absence of chromatographic separation. Recently, cyclic ion mobility mass spectrometry (cIM-MS) has been proposed to overcome this limitation. However, fluctuations in room conditions can affect ion mobility multipass arrival times, potentially reducing annotation confidence. In this study, we developed a multipass arrival time correction method that proved effective across various dates, room temperatures, ion mobility settings, and laboratories using mixtures of reference standards. We observed slight variations in the linear correction lines between lipid and nonlipid molecules, underscoring the importance of choosing appropriate reference molecules. Based on these results, we demonstrated that an accurate multipass arrival time database can be constructed from corrected t 0 and t p for interlaboratory use and can effectively identify isomeric lipids in MSI using only a single measurement. This approach significantly simplifies the identification process compared to determining multipass collision cross-section, which requires multiple measurements that are both sample- and time-intensive for MSI. Additionally, we validated our multipass drift time correction method in shotgun lipidomics analyses of human and mouse serum samples and observed no matrix effect for the analysis. Despite variations in dates, room temperatures, instruments, and ion mobility settings, our approach reduced the mean drift time differences from over 2% to below 0.2%.
    DOI:  https://doi.org/10.1021/acsmeasuresciau.4c00077
  7. Annu Rev Anal Chem (Palo Alto Calif). 2025 Feb 27.
    Machine Learning in Small-Molecule Mass Spectrometry.
    Yuhui Hong, Yuzhen Ye, Haixu Tang.
      Tandem mass spectrometry (MS/MS) is crucial for small-molecule analysis; however, traditional computational methods are limited by incomplete reference libraries and complex data processing. Machine learning (ML) is transforming small-molecule mass spectrometry in three key directions: (a) predicting MS/MS spectra and related physicochemical properties to expand reference libraries, (b) improving spectral matching through automated pattern extraction, and (c) predicting molecular structures of compounds directly from their MS/MS spectra. We review ML approaches for molecular representations [descriptors, simplified molecular-input line-entry (SMILE) strings, and graphs] and MS/MS spectra representations (using binned vectors and peak lists) along with recent advances in spectra prediction, retention time, collision cross sections, and spectral matching. Finally, we discuss ML-integrated workflows for chemical formula identification. By addressing the limitations of current methods for compound identification, these ML approaches can greatly enhance the understanding of biological processes and the development of diagnostic and therapeutic tools.
    DOI:  https://doi.org/10.1146/annurev-anchem-071224-082157
  8. Anal Chem. 2025 Feb 27.
    Human Untargeted Metabolomics in High-Throughput Gut Microbiome Research: Ethanol vs Methanol.
    Simone Zuffa, Vincent Charron-Lamoureux, Caitriona Brennan, Madison Ambre, Rob Knight, Pieter C Dorrestein.
      Untargeted metabolomics is frequently performed on human fecal samples in conjunction with sequencing to unravel the gut microbiome functionality. As sample collection efforts are rapidly expanding, with individuals often collecting specimens at home, metabolomics experiments should adapt to accommodate the safety and needs of bulk off-site collections and improve high throughput. Here, we show that a 95% ethanol, safe to be shipped and handled, extraction part of the Matrix Method pipeline recovers comparable amounts of metabolites as a validated 50% methanol extraction, preserving metabolic profile differences between investigated subjects. Additionally, we show that the fecal metabolome remains relatively stable when stored in 95% ethanol for up to 1 week at room temperature. Finally, we suggest a metabolomics data analysis workflow based on robust centered log ratio transformation, which removes the variance introduced by possible different sample weights and concentrations, allowing for reliable and integration-ready untargeted metabolomics experiments in gut microbiome research.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05142
  9. Curr Protoc. 2025 Feb;5(2): e70110
    Quantification of Glycosaminoglycans in Urine by Isotope Dilution Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry.
    Haoyue Zhang, James Beasley, Iskren Menkovic, Ashlee Stiles, David S Millington, Sarah P Young.
      Mucopolysaccharidoses (MPSs) are complex lysosomal diseases that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic/iduronic acid-N-acetylhexosamine or uronic/iduronic acid-N-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2025 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable-isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of methanolysis time Support Protocol 5: Measurement of methanolic HCl concentration Support Protocol 6: Preparation of working methanolic HCl solution (1.1 M) Support Protocol 7: Dilution of prepared urine sample.
    Keywords:  LC‐ESI‐MS/MS; dermatan sulfate; glycosaminoglycan; heparan sulfate; isotope dilution; mucopolysaccharidosis
    DOI:  https://doi.org/10.1002/cpz1.70110
  10. Anal Bioanal Chem. 2025 Feb 27.
    Integrating the lactulose-mannitol test for intestinal permeability with untargeted metabolomics for drug monitoring through dual liquid chromatography-mass spectrometry.
    Felina Hildebrand, Cemre Cukaci, Harald Schoeny, Christoph Baumgartinger, Bruno Stelzer, Matteo Spedicato, Tobias Frey, Martina Catani, Klaus Schmetterer, Richard Frey, Gunda Koellensperger.
      In this study, we developed a customized high-resolution mass spectrometry metabolomics workflow integrating the dual sugar test employing lactulose and mannitol as test probes for intestinal permeability assessment with untargeted screening of small molecules. Urine samples were collected from patients with major depression and healthy controls as part of a clinical study at the psychiatric department. Using a dual injection/dual chromatography setup, the test probes were quantified by hydrophilic interaction liquid chromatography (HILIC) in a targeted assay, while drugs and their metabolites were profiled in an untargeted manner by reversed-phase separation. Rigorous method development and validation allowed for selective separation of sugar isomers and consequently accurate quantification of lactulose and mannitol in urine. Internal standardization with compound specific stable isotope-labeled standards enabled excellent analytical figures of merit such as high recoveries, precision (< 5%), and working range (5 orders of magnitude). Within one analytical run, intestinal permeability was assessed together with drugs and their metabolites, allowing to screen for confounding drugs and patient compliance to the therapeutic scheme.
    Keywords:  Clinical/biomedical analysis; Drug monitoring; Dual LC–MS; Dual sugar test; Intestinal permeability testing
    DOI:  https://doi.org/10.1007/s00216-025-05790-7
  11. Talanta. 2025 Feb 18. pii: S0039-9140(25)00260-7. [Epub ahead of print]290 127770
    Improving the detectability of low-abundance p-cresol in biological matrices by chemical derivatization and LC-MS/MS determination.
    David Fabregat-Safont, Lena Coadic, Noemí Haro, Montserrat Montané-Garcia, Juliette Canaguier, Geoffroy Mallaret, Laetitia Davidovic, Óscar J Pozo.
      Gut microbiota produces a wide range of microbial metabolites with potential neuroactive properties. Among these, p-cresol, a by-product of tyrosine breakdown, has gained significant attention in various neuropsychiatric disorders, including autism spectrum disorder. However, current methods fail to detect p-cresol at trace levels in both the systemic circulation and brain, limiting the study of its role in neuropsychiatric disorders. There, the precise and accurate determination of p-cresol at low picogram levels is an unmet analytical need. To address this gap, we developed a highly-sensitive, validated method for quantifying p-cresol at low picogram levels in urine, plasma, and brain using chemical derivatization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that derivatization with 1,2-dimethylimidazole-5-sulfonyl chloride (5-DMIS-Cl or 5-DMISC) increased up to 40-fold the sensitivity compared to traditional dansyl derivatization. Therefore, a method based on 5-DMISC derivatization and sum of transitions was selected for validation. The method was accurate (recoveries 91-100 %) and precise (RSD <15 %) in all tested matrices, enabling detection down to100 pg/mL for urine, 20 pg/mL for plasma, and 0.04 pg/mg for brain tissue. The method was applied to plasma and brain samples from control and p-cresol-treated mice, revealing significant increases in p-cresol levels in treated animals. For the first time, we successfully quantified p-cresol levels in the brain, demonstrating its ability to cross the blood-brain barrier. In summary, this validated method offers a powerful tool for exploring the role of p-cresol -and potentially other phenolic compounds-in the microbiota-gut-brain axis and neuropsychiatric disorders.
    Keywords:  5-DMISC; Derivatization; Liquid chromatography; Mass spectrometry; Microbiota-gut-brain axis; p-Cresol
    DOI:  https://doi.org/10.1016/j.talanta.2025.127770
  12. Molecules. 2025 Feb 07. pii: 775. [Epub ahead of print]30(4):
    Determination of 14 Benzodiazepine Multiresidues in Aquaculture Environment by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
    Hongyang Guo, Jianwu Chen, Guangjun Jiang, Yuqing Mei, Zhiqiang Gong, Mingdian Liu, Jinping Li, Jinhua Gan.
      In this study, an analytical method for the simultaneous determination of 14 benzodiazepine (BDZ) multiresidues in aquaculture environmental water and sediment was developed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The method uses an internal standard for quantification and achieves chromatographic separation and analysis within 11 min. The results of method validation showed that the recoveries of most analytes were in the range of 70-120% in water or sediment matrices, and the correlation coefficients of the 14 target chemistries were R2 > 0.99, with relative standard deviations (RSD) < 15%. The limits of detection (LODs) and the limits of quantification (LOQs) were in the ranges of 0.002-0.01 μg/L and 0.01-0.03 μg/L for water and 0.01-0.5 μg/kg and 0.04-1 μg/kg for the sediment matrix. The method is simple and has high rapidity, high sensitivity, and low cost. It provides technical support for the simultaneous monitoring of BDZ residues in the aquaculture environment.
    Keywords:  aquaculture environment; benzodiazepines; multiresidues; ultra-high-performance liquid chromatography–tandem mass spectrometry
    DOI:  https://doi.org/10.3390/molecules30040775
  13. Molecules. 2025 Feb 08. pii: 782. [Epub ahead of print]30(4):
    Validation of an LC-HRMS Method for Quantifying Indoxyl Sulfate and p-Cresyl Sulfate in Human Serum.
    María Rodríguez-García, Irene Martínez, Irene Aliart, Jaime I Sainz de Medrano, Nayra Rico, Víctor Joaquín Escudero-Saiz, Francisco Maduell, Manuel Morales-Ruiz, Gregori Casals.
      Accurate quantification of indoxyl sulfate (IndS) and p-cresyl sulfate (pCS) is essential for understanding their role in chronic kidney disease (CKD) progression and for developing strategies to mitigate their harmful effects, including cardiovascular morbidity and renal fibrosis. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) enable the integration of powerful diagnostic tools into clinical laboratories. Along with accurate quantification, precise mass measurements allow for untargeted compound identification.
    METHODS: An LC-HRMS was validated for quantifying IndS and pCS in human serum, following EMA guidelines. The method involved protein precipitation with methanol, micro-LC for chromatographic separation, and detection based on accurate mass, with simultaneous high-resolution full-scan acquisition. Clinical samples from patients with varying degrees of renal insufficiency and samples obtained before and after hemodiafiltration were analyzed.
    RESULTS: The method demonstrated acceptable linearity, precision, and accuracy. The measurement range for both analytes was from 100 to 40,000 ng/mL. Serum levels of IndS and pCS correlated with decreased renal function. After hemodiafiltration, there was a significant reduction of IndS (50%) and pCS (43%). Simultaneous untargeted analysis allowed to identify metabolites significantly underexpressed after hemodiafiltration.
    CONCLUSIONS: An accurate LC-HRMS method was validated for the quantification of IndS and pCS serum levels in patients with CKD, providing insights into toxin dynamics and enabling untargeted metabolic evaluation.
    Keywords:  chronic kidney disease; hemodiafiltration; high-resolution mass spectrometry; metabolomics; micro-liquid chromatography; uremic toxins
    DOI:  https://doi.org/10.3390/molecules30040782
  14. Anal Biochem. 2025 Feb 24. pii: S0003-2697(25)00059-4. [Epub ahead of print] 115821
    Simultaneous determination of 25(OH)D2, 25(OH)D3 and 1α,25(OH)2D3 in human serum by derivatization-liquid chromatography-tandem mass spectrometry.
    Zi-Qing Wang, Li-Ping Hao, Zi-Xuan Meng, Hao-Ran Zhang, Wei-Jun Kang, Lian-Feng Ai.
      An LC-MS/MS method was developed and validated for simultaneously quantifying 25-hydroxyvitamin D2, D3, and 1α,25-dihydroxyvitamin D3 in human serum. Protein in 200 uL serum was precipitated with acetonitrile. After centrifugation, the metabolites were derivatised using (diacetoxyiodo)benzene (PIA) and 4-(4-dimethylaminophenyl)-1,2,4-triazolidine-3,5-dione (DMAT) and then quantified by the LC-MS/MS system. The limits of detection (LODs) for the three substances were 10, 10, and 5 pg/mL, and the limits of quantification (LOQs) were 20, 20, and 10 pg/mL. The standard curves for these compounds showed linear regression coefficients (R2)>0.998 over specific concentration ranges. Recoveries were 94.36% - 102.34% for 25(OH)D2, 92.43% - 103.41% for 25(OH)D3, and 88.98% - 94.36% for 1α,25(OH)2D3. The mean serum levels in 109 subjects (consisting of 61 healthy adult males and 59 healthy adult females) were 2.0 ± 1.5 ng/ml (25(OH)D2), 16.4 ± 6.1 ng/ml (25(OH)D3) and 36.6 ± 15.1 pg/ml (1α, 25(OH)2D3).Derivatization of vitamin D metabolites using PIA and DMAT is useful for rapidly determining the serum 25(OH) D2, 25(OH) D3 and 1α,25(OH)2D3 concentrations simultaneously in human serum.
    Keywords:  1α,25-dihydroxyvitamin D3; 25-Hydroxyvitamin D; Derivatization; liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.ab.2025.115821
  15. ACS Meas Sci Au. 2025 Feb 19. 5(1): 135-144
    Challenges in PFAS Postdegradation Analysis: Insights from the PFAS-CTAB Model System.
    Chanaka Navarathna, Ransford Appianin Boateng, Long Luo.
      Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals widely used for their oil and water-repellent properties. Their environmental persistence and potential health risks have raised significant concerns. As PFAS degrades through remediation or natural processes, they form complex mixtures of the original chemicals, transformation byproducts, and degradation additives. Analyzing PFAS after degradation presents analytical challenges due to possible chemical and physical interactions, including ion pairing, micelle formation, and complexation. These factors can significantly impact the precision and accuracy of PFAS measurements, yet they are often overlooked in PFAS degradation studies. In this work, we demonstrate that with the addition of ppb-level cetyltrimethylammonium bromide (CTAB), a cationic surfactant used in PFAS plasma-based degradation, the PFAS calibration curve linearity, sensitivity, and reproducibility are severely compromised. Isotopically labeled internal standards cannot fully correct these issues. Furthermore, the standard EPA methods 537.1, 533, and 1633 could not accurately recover PFAS concentrations in the PFAS and CTAB mixtures, with severe matrix effects observed for longer-chain and nitrogen-containing PFAS. Among these methods, Method 1633 is currently the most suitable option for postdegradation analysis. Method 1633 showed the lowest CTAB interference because this method used another weak ion pair additive, formic acid or acetic acid (in commercial lab analysis), to acidify the sample before LC-MS/MS analysis and added an isotopically labeled internal standard. For future PFAS degradation studies, we recommend systematically evaluating the matrix effect on the PFAS quantification using a recovery matrix to validate the analytical methods before use.
    DOI:  https://doi.org/10.1021/acsmeasuresciau.4c00083
  16. Talanta. 2025 Feb 08. pii: S0039-9140(25)00198-5. [Epub ahead of print]289 127712
    Comprehensive analysis of oxidized arachidonoyl-containing glycerophosphocholines using ion mobility spectrometry-mass spectrometry.
    Sandra M Camunas-Alberca, Ameer Y Taha, Ana Gradillas, Coral Barbas.
      The biological significance of oxidized arachidonoyl-containing glycerophosphocholines, exemplified by the oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), in pathological processes is well-established. However, despite their widespread use in redox lipidomics research, the precise chemical composition of the heterogeneous mixtures of oxPAPC generated in vitro -including the high prevalence of isomers and the oxidation mechanisms involved- remain inadequately understood. To address these knowledge gaps, we developed a multidimensional in-house database from a commercial oxPAPC preparation -employing Liquid Chromatography coupled to Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS) and Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS). This database includes lipid names, retention times, accurate mass values (m/z), adduct profiles, MS/MS information, as well as collision cross-section (CCS) values. Our investigation elucidated 34 compounds belonging to distinct subsets of oxPAPC products, encompassing truncated, full-length, and cyclized variants. The integration of IMS-MS crucially facilitated: (i) structural insights among regioisomers, exemplified by the 5,6-PEIPC and 11,12-PEIPC epoxy-isoprostane derivatives, (ii) novel Collision Cross Section (CCS) values, and (iii) cleaner MS/MS spectra for elucidating the fragmentation mechanisms involved to yield specific fragment ions. These diagnostic ions were employed to successfully characterize full-length isomers present in human plasma samples from patients with mucormycosis. This comprehensive oxPAPC characterization not only advances the understanding of lipid peroxidation products but also enhances analytical capabilities for in vitro-generated oxidized mixtures. The implementation of this robust database, containing multiple orthogonal (i.e., independent) pieces of information, will serve as a comprehensive resource for the field.
    Keywords:  Arachidonic Acid (AA); Ion Mobility Spectrometry (IMS); Mass Spectrometry (MS); Multidimensional LC-MS Database; Oxidized Glycerophosphocholines; Redox Lipidomics; oxPAPC
    DOI:  https://doi.org/10.1016/j.talanta.2025.127712
  17. Metabolites. 2025 Feb 08. pii: 110. [Epub ahead of print]15(2):
    1H-NMR Lipidomics, Comparing Fatty Acids and Lipids in Cow, Goat, Almond, Cashew, Soy, and Coconut Milk Using NMR and Mass Spectrometry.
    Brianna Williams, Shamika P W R Hewage, Denzel Alexander, Harshica Fernando.
      Background/Objectives: Lipids are an important component of human nutrition. Conventional milk is obtained from animals, and dairy milk is consumed by many people worldwide. Recently, milk consumers have been increasingly shifting towards plant-based milk options. The aim of the study was the qualitative identification of lipid metabolites in animal- and plant-based milk, the identification and comparison of the fatty acids (FAs) of milk, and the qualitative identification of the lipid groups among the milk varieties. Methods: Milk samples were obtained from local grocery stores. Lipids were extracted using a modified Folch method and analyzed using nuclear magnetic resonance (NMR) metabolomics. Gas and liquid chromatography mass spectrometry methods (GC-MS and LC-MS) were used to identify the FAs and lipid groups. Lipid weights were compared and the NMR profiles of the lipids analyzed by multivariate statistical analysis. Principal component analysis was performed for the milk lipids obtained from the animal, and plant milk varieties. Results: Clustering of NMR data showed two main clusters: cow/almond/cashew and goat/soy/coconut. GC-MS analysis of the methylated fatty acids (FAs) showed the presence of 12:0, 14:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, 20:1, and 20:2 in all milk types, while FAs 19:0 and 20:4 were observed only in the dairy milk. LC-MS data showed common masses that may indicate the presence of mono- and diacyl glycerols and several lysophospholipids among the different types of milk. Conclusions: This study shows the advantage of using NMR, GC-MS, and LC-MS to differentiate the lipids among different milk types and compare them on one platform.
    Keywords:  animal milk; fats; fatty acids; lipids; phospholipids; plant milk
    DOI:  https://doi.org/10.3390/metabo15020110
  18. Anal Chem. 2025 Feb 26.
    Structural Annotation Method for Locating sn- and C═C Positions of Lipids Using Liquid Chromatography-Electron Impact Excitation of Ions from Organics (EIEIO)-Mass Spectrometry.
    Yao Chen, Jun Yang, Xinxin Wang, Yuqing Zhang, Yaping Shao, Hang Li, Xiaoyan Dong, Fei Jiang, Chunxiu Hu, Guowang Xu.
      Definitive structural elucidation of lipids is pivotal for unraveling the functions of lipids in biological systems. Despite advancements in mass spectrometry (MS) for lipid analysis, challenges in annotation scope and efficiency remain, especially in resolving isomers. Herein, we introduce an optimized method using liquid chromatography coupled with electron impact excitation of ions from organic tandem mass spectrometry (LC-EIEIO-MS/MS) for comprehensive analysis and structural annotation of lipids. This approach integrates a six-step analytical protocol for precise lipid annotation, including (1) extracting MS information, (2) classifying lipids, (3) aligning sum composition, (4) determining sn-positions, (5) locating C═C positions, and (6) ascertaining annotation levels. In analyzing 34 lipid standards spiked into serum, our method achieved 100% and 82.4% annotation accuracy at the sn- and C═C isomer levels, respectively, compared to 26.5% and 0% in the CID mode using MS-DIAL. A total of 1312 sn-positions and 1033 C═C locations of lipids were annotated in quality control plasma pooled from healthy individuals and patients with Alzheimer's disease. The isomers of lipids revealed more pronounced differences between the healthy and diseased groups compared to the sum compositions of the lipids. Overall, the LC-EIEIO-MS/MS approach provides a comprehensive profiling and efficient annotation method for lipidomics, promising to shed new light on lipid-related biological pathways and disease mechanisms.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05560
  19. Anal Chim Acta. 2025 Apr 01. pii: S0003-2670(25)00132-1. [Epub ahead of print]1345 343738
    Nanoconfined in-situ sampling and extracting switching needle device construction for highly enrichment of polar metabolites in human urine.
    Chunyu Yu, Yifan Jiao, Lang Bi, Yilin Zou, Jinhua Zhao, Meihui Tong, Xiaohan Zong, Jiaxin Zhang, Songnan Zhang, Donghao Li.
       BACKGROUND: Urine, as a non-invasive sampling specimen, offers significant advantages for the diagnosis and treatment of diseases. However, as a water-based matrix, most metabolites in urine are high polarity, which limits the ultrafast extraction and high enrichment of these polar metabolites from urine. Though solvent-based extraction method has demonstrated considerable benefits in extraction rates, the necessity for selecting water-immiscible solvents restricts the extraction of highly polar metabolites. Consequently, there is a pressing need for a rapid extraction method that achieves a high enrichment factor specifically targeting highly polar metabolites in urine. (90) RESULTS: This study is the first attempt to use water-soluble solvent to extract highly polar metabolites from urine. Firstly, a needle device capable of switching between sampling and extracting modes was constructed by utilizing carbon nanofibers/carbon fibers (CNFs/CFs) filled with the needle tip. Under the effect of nanoconfinement, the needle can achieve in-situ ultrafast extraction employing a methanol/water mixture as the nanoconfined extraction solvent. Additionally, the nanodistribution of the extracted solvent within the nanoconfined space enhances the exaction rate and efficiency. By coupling this method with HILIC-LC/MS detection protocol, 33 highly polar metabolites from human urine were simultaneously quantified within 3 min. Following systematic validation, the established method was successfully applied to urine target metabolomics analysis for hepatocellular carcinoma, the potential diagnosis biomarkers were screened out using multiple data processing methods. (130) SIGNIFICANCE: In conclusion, the home-made nanoconfinement effect based in-situ sampling and extracting (NISE) switching needle device demonstrates strong advantages in the highly enrichment and ultrafast analysis of highly polar metabolites in an aqueous matrix, providing a promising tool for clinical disease diagnosis and screening. (44).
    Keywords:  Extraction and enrichment; Hepatocellular carcinoma; In-situ sampling and extracting; Nanoconfinement; Urine metabolomics
    DOI:  https://doi.org/10.1016/j.aca.2025.343738
  20. Metabolites. 2025 Feb 19. pii: 140. [Epub ahead of print]15(2):
    Matrix Linear Models for Connecting Metabolite Composition to Individual Characteristics.
    Gregory Farage, Chenhao Zhao, Hyo Young Choi, Timothy J Garrett, Marshall B Elam, Katerina Kechris, Śaunak Sen.
      Background/Objectives: High-throughput metabolomics data provide a detailed molecular window into biological processes. We consider the problem of assessing how association of metabolite levels with individual (sample) characteristics, such as sex or treatment, depend on metabolite characteristics such as pathways. Typically, this is done using a two-step process. In the first step, we assess the association of each metabolite with individual characteristics. In the second step, an enrichment analysis is performed by metabolite characteristics. Methods: We combine the two steps using a bilinear model based on the matrix linear model (MLM) framework previously developed for high-throughput genetic screens. Our method can estimate relationships in metabolites sharing known characteristics, whether categorical (such as type of lipid or pathway) or numerical (such as number of double bonds in triglycerides). Results: We demonstrate the flexibility and interoperability of MLMs by applying them to three metabolomic studies. We show that our approach can separate the contribution of the overlapping triglyceride characteristics, such as the number of double bonds and the number of carbon atoms. Conclusion: The matrix linear model offers a flexible, efficient, and interpretable framework for integrating external information and examining complex relationships in metabolomics data. Our method has been implemented in the open-source Julia package, MatrixLM. Data analysis scripts with example data analyses are also available.
    Keywords:  Julia language; bilinear models; high-throughput data; lipidomics; metabolomics
    DOI:  https://doi.org/10.3390/metabo15020140
  21. Anal Chem. 2025 Feb 28.
    Enhancing the Accuracy of Identification and Relative Quantification of Unsaturated Fatty Acids in Serum via a Stable Isotope-Labeled Double Derivatization Strategy.
    Yunxia Yuan, Bingqing Chi, Jiajia Fan, Ying Wang, Gan Luo, Xiaoyan Gao.
      Accurate identification and quantification of fatty acids are critical for investigating their biological function in disease models. Although several derivatization methods have been proposed for identifying the positions of C═C bonds in unsaturated fatty acids, poor ionization efficiency of the carboxyl group leads to lower intensity of molecular ion peaks, making their identification difficult and interfering with the accuracy of quantification based on peak areas of characteristic ion pairs. In this study, a strategy of stable isotope-labeled carboxyl derivatization combined with C═C derivatization was employed for simultaneously the identification and quantification of fatty acids using d0/d9-5-amino-N,N,N-trimethylpentane-1-ammonium iodide (d0/d9-ATPAI) to label the carboxyl group and m-chloroperoxybenzoic acid to label C═C bonds. The stable isotope-labeled quaternary amine groups in the novel carboxyl derivatization reagent d0/d9-ATPAI can enhance the accuracy of the recognition of characteristic ion pairs to facilitate the structural elucidation of various fatty acids. The heavy isotope-labeled fatty acids can be served as internal standards to achieve accurate relative quantification of the C═C position isomers of individual unsaturated fatty acids among samples based on the peak area ratio of the characteristic ion pairs. Unsaturated fatty acid C═C positional isomers were quantified using aldehyde or alkenyl diagnostic ions. In addition, saturated fatty acids were quantified using the m/z 86.09679 cyclamine characteristic ion. This approach enhanced the detection sensitivity of fatty acids by 60,000 times, allowing for the characterization of 70 fatty acids in rat serum, including 26 unsaturated fatty acid C═C positional isomers. Pseudotargeted metabolomics analysis of serum fatty acids revealed alterations in the fatty acid metabolic pathway during diabetic cognitive dysfunction. Overall, the proposed method, with high sensitivity and wide coverage, could provide accurate identification and relative quantification of various fatty acids in complex matrices.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06375
  22. Front Pharmacol. 2025 ;16 1544215
    UPLC-MS/MS method for simultaneous determination of pyrantel, praziquantel, febantel, fenbendazole and oxfendazole in dog plasma and its application to a bioequivalence study.
    Fengyichi Zhang, Fanxi Guo, Xinyu Zhao, Chongyang Li, Junli Wang, Mengyun Wang, Ruixia Ma, Baochang Chen, Qiangqiang Miao, Yimeng Wang, Zihan Wang, Di Cao, Zugong Yu.
      A rapid, sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the simultaneous quantitation of pyrantel (PYR), praziquantel (PRA), febantel (FBT) and its active metabolites, fenbendazole (FEN) and oxfendazole (OXF) in dog plasma. Fenbendazole-D3 (FEN-D3) was used as an internal standard (IS). The analytes and IS were prepared using simple protein precipitation (PP) combined with liquid-liquid extraction (LLE). Chromatographic analysis was performed on UPLC BEH C18 column using acetonitrile-0.1% formic acid in water for gradient elution. Detection was carried out in multiple reaction monitoring (MRM) mode under positive electrospray ionization. The standard curves were linear through the concentration range of 4-240 ng/mL for PYR and OXF, 15-900 ng/mL for PRA, 2-120 ng/mL for FBT and 10-600 ng/mL for FEN with all correlation coefficients >0.99. The intra-and inter-batch precision was 1.08%-14.26% and accuracy was from 90.66% to 110.28%. The mean extraction recoveries for the analytes and IS were >90%. The total run time was 9.0 min. The developed method was successfully applied to a bioequivalence study after oral administration of compound febantel tablets in 38 healthy dogs.
    Keywords:  UPLC-MS/MS; bioequivalence; dog plasma concentration; febantel; fenbendazole; oxfendazole; praziquantel; pyrantel
    DOI:  https://doi.org/10.3389/fphar.2025.1544215
  23. Analyst. 2025 Feb 25.
    Evaluating sample normalization methods for MS-based multi-omics and the application to a neurodegenerative mouse model.
    Gwang Bin Lee, Cha Yang, Fenghua Hu, Ling Hao.
      Mass spectrometry (MS)-based omics methods have transformed biomedical research with accurate and high-throughput analysis of diverse molecules in biological systems. Recent technological advances also enabled multi-omics to be achieved from the same sample or on a single analytical platform. Sample normalization is a critical step in MS-omics studies but is usually conducted independently for each omics experiment. To bridge this technical gap, we evaluated different sample normalization methods suitable for analyzing proteins, lipids, and metabolites from the same sample for multi-omics analysis. We found that normalizing samples based on tissue weight or protein concentration before or after extraction generated distinct quantitative results. Normalizing samples first by tissue weight before extraction and then by protein concentration after extraction resulted in the lowest sample variation to reveal true biological differences. We then applied this two-step normalization method to investigate multi-omics profiles of mouse brains lacking the GRN gene. Loss-of-function mutations in the GRN gene lead to the deficiency of the progranulin protein and eventually cause neurodegeneration. Comparing the proteomics, lipidomics, and metabolomics profiles of GRN KO and WT mouse brains revealed molecular changes and pathways related to lysosomal dysfunction and neuroinflammation. In summary, we demonstrated the importance of selecting an appropriate normalization method during multi-omics sample preparation. Our normalization method is applicable to all tissue-based multi-omics studies, ensuring reliable and accurate biomolecule quantification for biological comparisons.
    DOI:  https://doi.org/10.1039/d4an01573h
  24. Anal Chem. 2025 Feb 25.
    Microflow Liquid Chromatography Coupled to Multinozzle Electrospray Ionization for Improved Lipidomics Coverage of 3D Clear Cell Renal Cell Carcinoma.
    Sergey Girel, Mathieu Galmiche, Mathis Fiault, Valentin Mieville, Patrycja Nowak-Sliwinska, Serge Rudaz, Isabel Meister.
      In most bioanalytical laboratories, high-resolution mass spectrometry (HRMS) systems with electrospray ionization (ESI) are hyphenated to liquid chromatography platforms. The latter typically operate under analytical flow (AF; 0.2-1 mL/min) regimes. Hence, AF/ESI-HRMS methods prioritize the detection of analytes of higher abundances or ionizability and tend to suffer from matrix effects or ion suppression. A far higher sensitivity can be obtained with electrospray at nanoflow (10-1000 nL/min) thanks to a better ionization efficiency and significant decrease in matrix effects. Both advantages are crucial to reliably accessing low-abundance compounds or weakly ionizable analytes. This work presents a microflow (μF) chromatographic setup coupled to a novel microfabricated multinozzle electrospray (mnESI) emitter with five nozzles spraying at 600 nL/min per nozzle for untargeted HRMS lipidomic profiling. With a runtime of 19 min, similar to our established analytical flow (AF/ESI) lipidomics platform, μF/mnESI produced a 16-fold median increase across 69 deuterated lipid standards. The performance of this new configuration was also evaluated in the context of the profiling of a 3D clear cell renal cell carcinoma (ccRCC) model exposed to a multidrug combination therapy. The processing of the acquired data resulted in 1270 (μF/mnESI) vs 752 (AF/ESI) MS2-annotated lipids. Among those, 762 achieved <10% variation on pooled QC samples for μF/mnESI compared to only 361 for the AF method. In addition, the measurements of ccRCC samples confirmed the improvements in ionization efficiency and adduct patterns observed with standards, enabling to annotate 79 oxidized triglycerides, 38 cholesterol esters (only five and four detected in AF/ESI, respectively), and 12 sitosterol esters, not yet reported in mammalian cell cultures.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06337
  25. Molecules. 2025 Feb 07. pii: 758. [Epub ahead of print]30(4):
    A Systematic Study of Liquid Chromatography in Search of the Best Separation of Cannabinoids for Potency Testing of Hemp-Based Products.
    Ayowole Owolabi, Olalekan Ogunsola, Emma Joens, Medline Kotler, Liguo Song.
      A study was conducted to search for the best separation of eighteen cannabinoids, the maximum number of cannabinoids that have been quantified so far, for potency testing of hemp-based products using liquid chromatography diode array detector (LC-DAD). The investigation utilized four column types, all sharing the same dimension (150 mm × 2.1 mm) and core-shell particle size (2.7 µm), but different stationary phases: dimethyl-octadecyl (Poroshell 120 EC-C18), diisobutyl-octadecyl (Raptor ARC-18), reverse phase (RP)-carbamate (Cortecs Shield RP-18), and RP-amide (Ascentis Express RP-Amide). The resolution of adjacent cannabinoids was kept close to 1.5 or higher, while the separation time was kept as short as possible. The fastest separation was achieved within 15.0 min using two sequential Raptor ARC-18 columns, with a mobile phase consisting of 75.0% acetonitrile and 25.0% aqueous solution of 0.03% formic acid and 0.5 mM ammonium formate at pH 2.97, at a flow rate of 0.5 mL/min. A slightly improved resolution of the eighteen cannabinoids was obtained within 18.5 min using two sequential Poroshell 120 EC-C18 columns under similar conditions, except for a mobile phase containing 77.5% acetonitrile and a reduced flow rate of 0.45 mL/min due to backpressure higher than 600 bars. Furthermore, a rapid 7.0 min separation was achieved for potency testing of hemp-based products by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) using a Cortecs Shield RP-18 column, with a mobile phase consisting of 70.0% acetonitrile and 30.0% aqueous solution of 0.01% formic acid and 1 mM ammonium formate at pH 3.38 at a flow rate of 0.5 mL/min.
    Keywords:  cannabinoids; chromatographic separation; hemp; potency testing; stationary phase
    DOI:  https://doi.org/10.3390/molecules30040758
  26. Molecules. 2025 Feb 12. pii: 852. [Epub ahead of print]30(4):
    Simultaneous Determination of Six Acidic Herbicides and Metabolites in Plant Origin Matrices by QuEChERS-UPLC-MS/MS.
    Qiqi Jin, Qianwen Xu, Zhiyong Zhao, Wenshuai Si, Bing Bai, Lei Chen, Changyan Zhou.
      This study presents a method for the simultaneous determination of six acidic herbicides and their metabolites in various matrices, including fruits, vegetables, grains, and edible oils. The method employs acidified acetonitrile extraction combined with dispersive solid-phase extraction cleanup (dSPE) using MgSO4, Florisil, and Graphitized carbon black (GCB). The analysis was performed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with electrospray ionization (ESI) in both positive and negative modes using multiple reaction monitoring (MRM). The mass concentrations of six herbicide pesticides and their metabolites were predominantly within the range of 0.0005~0.050 mg/L and exhibited strong linear relationships with the corresponding peak area, with the coefficient of determination (R2) exceeding 0.993. The limits of detection (LOD) for the method ranged from 0.0001 to 0.008 mg/kg. The recovery rates of adding recovery experiments to cabbage, chives, pear, wheat flour, and soybean oil were 69.8~120%, and the relative standard deviation (RSD) was 0.6~19.5%. The results indicate that this method is efficient and fast, and can be used for the detection of compounds in various actual matrices.
    Keywords:  QuEChERS; UPLC-MS/MS; acid herbicides; residue detection
    DOI:  https://doi.org/10.3390/molecules30040852
  27. Biomed Chromatogr. 2025 Apr;39(4): e70033
    Pharmacokinetic Study of HRO761 in Rats by Liquid Chromatography Combined With Electrospray Ionization Tandem Mass Spectrometry.
    Jie Shen, Liye Ge, Guowei Jiang, Yongjun Meng, Weina Ma.
      A simple and sensitive liquid chromatography tandem mass spectrometry method was established and validated for determination of HRO761 in rat plasma. After prepared by protein precipitation with acetonitrile, HRO761 and internal standard were separated on a Waters BEH C18 column using acetonitrile containing 0.1% formic acid and 0.1% formic acid in water as mobile phase by gradient elution. The method showed excellent linearity over the range of 5-5000 ng/mL with acceptable intraday and interday precision, accuracy, matrix effect, and recovery. The stability assay indicated that HRO761 was stable during the sample acquisition, preparation, and storage. The method was applied to pharmacokinetic study of HRO761 in rats. The result suggested that after intravenous administration at dose of 1 mg/kg, HRO761 was quickly eliminated from the plasma with the elimination half-life of 1.9 h. After oral administration at doses of 5, 10, and 20 mg/kg, HRO761 was quickly absorbed into plasma and reach the peak concentration (Cmax) of 2598.1-9379.2 ng/mL at 1.0-4.0 h. The exposure increased proportionally with the dose. The oral bioavailability was 79.0%-99.1% over the range of 5-20 mg/kg. This study provides useful information for its further development in clinic.
    Keywords:  HRO761; LC–MS/MS; bioavailability; pharmacokinetic
    DOI:  https://doi.org/10.1002/bmc.70033
  28. J Chromatogr A. 2025 Feb 19. pii: S0021-9673(25)00150-5. [Epub ahead of print]1746 465802
    Rapid single-step pretreatment of urine samples using a 2D carbon microfiber fractionation for phthalate and metabolite detection via GC-MS and LC-MS/MS.
    Lei Yang, Lian Hong, Yu Qiu, Meiyu Cui, Donatella Nardiello, Maurizio Quinto, Chunyu Yu, Jinhua Zhao, Donghao Li.
      Phthalates (PAEs) are widely used as plasticizers and softeners. PAEs and their metabolites (PMs) are considered toxic substances, causing endocrine disruption and damage to reproductive and immune systems. Due to their different polarities, a comprehensive analysis of PAEs and PMs in real samples requires multi-step, complex, and expensive pretreatments, increasing the possibility of sample contamination or analyte losses. In this work, a fast and eco-sustainable sample pretreatment method that allows PAEs and PMs analysis in urine samples by using a two-dimensional microscale micro-carbon fiber (CF) / active micro-carbon fiber (ACF) (2DµCFs) device is proposed. 2DµCFs device allows PAEs, PMs, and water-soluble impurities separation in a single pretreatment exploiting the selective adsorption of CFs and ACFs that is carried out by using small solvent amounts. Furthermore, the 2DµCFs device reduces the matrix effect allowing reliable GC-MS and LC-MS/MS analyses. Limits of quantification were in the range of 15-25 ng mL-1 and of 20-30 ng mL-1, and recoveries were in the range of 78.1 %-87.1 % and 79.4 %-97.3 %, for PAEs and PMs, respectively. The method also showed good reproducibility, with relative standard deviations in the range of 3.8 %-13.3 % and 3.3 %-11.3 % for PAEs and PMs, respectively. Considering these results, the 2DµCFs sample pretreatment procedure can be considered a powerful tool for effective, fast, accurate, and eco-sustainable PAEs and PMs analyses in real samples.
    Keywords:  Carbon fiber; Phthalates; Phthalates metabolites; Sample pretreatment
    DOI:  https://doi.org/10.1016/j.chroma.2025.465802
  29. Metabolites. 2025 Feb 07. pii: 106. [Epub ahead of print]15(2):
    Reference Intervals of Serum Metabolites and Lipids of a Healthy Chinese Population Determined by Liquid Chromatography-Mass Spectrometry.
    Yuqing Zhang, Jinhui Zhao, Hui Zhao, Xin Lu, Xueni Jia, Xinjie Zhao, Guowang Xu.
      Background: Metabolomics serves as a very useful tool for elucidating disease mechanisms and identifying biomarkers. Establishing reference intervals (RIs) of metabolites in a healthy population is crucial to the application of metabolomics in life sciences and clinics. Methods: We enrolled 615 healthy Chinese adults aged between 21 and 85 years. Their health status was ascertained through clinical examinations, biochemical parameters, and medical history. Targeted metabolomics and lipidomics analyses were applied to quantify 705 metabolites and lipids in the serum, establishing RIs and investigating the effect of sex and age on the metabolome and lipidome. Results: This study is the first large-scale effort in China to establish RIs for metabolites in the apparently healthy population. We found that most of the sex-related metabolites, including amino acids, acyl-carnitines and triacylglycerols, had higher concentrations in males, while the other sex-related lipids showed higher concentrations in females. Most of the age-related metabolites increased with age, including those associated with protein synthesis, nitric oxide synthesis, energy metabolism, and lipid metabolism. Conclusions: This study gives the reference intervals of the healthy Chinese metabolome and lipidome and their relationship with sex and age, which facilitates life sciences and precision medicine, especially for disease research and biomarker discovery.
    Keywords:  lipidomics; liquid chromatography; mass spectrometry; metabolomics; quantitative analysis; reference intervals
    DOI:  https://doi.org/10.3390/metabo15020106
  30. J Steroid Biochem Mol Biol. 2025 Feb 21. pii: S0960-0760(25)00040-8. [Epub ahead of print] 106712
    Targeted Quantitative Analysis of Urinary Bile Acids by Liquid Chromatography-Tandem Mass Spectrometry: Method Development and Application to Healthy and Obese Children.
    M Schauermann, R Wang, J Pons-Kuehnemann, M F Hartmann, T Remer, Y Hua, A Bereket, M Wasniewska, M Shmoish, Z Hochberg, A Gawlik, S A Wudy.
      Bile acids (BA) are C24 steroids synthesized from cholesterol in liver. Hardly any data exist on BA in the most accessible human biofluid urine. As bile acids bear great potential as future biomarkers in diagnosis and monitoring of metabolic diseases, we aimed at developing and implementing a new method for the quantification of urinary bile acids using targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). A second goal consisted in creating first reference values of urinary bile acids during childhood and to investigate their excretion patterns in obese children and adolescents. Our method required 2mL of urine and sample preparation consisting of protein precipitation and solid phase extraction. Stable isotopes of BA were included as internal standards (IS). Our method is capable of simultaneously determining 18 BA: the primary BA cholic acid (CA) and chenodeoxycholic acid (CDCA), and the secondary BA deoxycholic acid (DCA) and lithocholic acid (LCA) as well as glycine and taurine conjugates of these four BA. Furthermore, ursodeoxycholic acid (UDCA) and five BA in their sulfated forms (LCA-S, GLCA-S, TLCA-S, GCDCA-S, GDCA-S) were analyzed. After successful validation (intra-day precision 1.02% - 11.07%; inter-day precision 0.42% to 11.47%.; intra-day accuracy 85.75% - 108.90%; inter-day accuracy 86.76% - 110.99%; no significant matrix effect; recovery 90.49% - 113.99%)., the method was applied to samples of 80 healthy children as well as 237 obese children of various age groups. Sulfated BA showed the highest concentrations, with GCDCA-S (nmol/L, medians, controls vs. obese 588.4 vs. 360.2) being the most abundant among all BA, followed by GLCA-S (353.9 vs. 344.8) and GDCA-S (319,3 vs. 323.9). CA (135.1 vs. 174.6) and GCA (100.2 vs. 92.4) were the two dominant non-sulfated BA. In conclusion, we developed a LC-MS/MS method for the simultaneous determination of 18 urinary bile acids in children and adolescents. We created reference values and investigated obese children. Sulfated bile acids dominated in both study groups. Lower bile acid sulfation and amidation in obese children point to limitations in their hepatic metabolic capacity.
    DOI:  https://doi.org/10.1016/j.jsbmb.2025.106712
  31. Curr Protoc. 2025 Feb;5(2): e70108
    Visualization of Efavirenz-Induced Lipid Alterations in the Mouse Brain Using MALDI Mass Spectrometry Imaging.
    Nav Raj Phulara, Herana Kamal Seneviratne.
      This article highlights experimental procedures and troubleshooting tips for the utilization of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) methods for detecting and visualizing lipid alterations in the mouse brain tissue in response to efavirenz (EFV) treatment. To investigate drug-induced adverse effects, it is becoming increasingly important to understand the spatial alterations of lipid molecules in the target organs. EFV is a non-nucleoside reverse transcriptase inhibitor commonly used for HIV treatment in combination with other antiretrovirals. Importantly, EFV is a drug that is included in the World Health Organization's list of essential medications. However, EFV is known to be associated with neurotoxicity. To date, the mechanisms underlying EFV-induced neurotoxicity have not been fully elucidated. Therefore, it is important to gain understanding of the effect of EFV on the brain. It is known that the brain is composed of different neuroanatomical regions that are abundant in lipids. Described here is the use of a chemical imaging strategy, MALDI MSI, to detect, identify, and visualize the spatial localization of several lipid species across the brain tissue sections along with their alterations in response to EFV treatment. The set of protocols consists of three major parts: lipid detection, identification, and tissue imaging. Lipid detection includes testing different chemical matrices and how they facilitate the detection of analytes, which is then followed by identification. Collision-induced dissociation is employed to verify the identity of the lipid molecules. Lastly, tissue imaging experiments are performed to generate the spatial localization profiles of the lipids. The protocols described in this article can be employed to spatially visualize alterations in the lipid molecules in response to drug treatment. © 2025 Wiley Periodicals LLC. Basic Protocol 1: MALDI mass spectrometry (MALDI MS) profiling experiments for detection of lipids Basic Protocol 2: MALDI MS imaging of lipid molecules in mouse brain tissues Basic Protocol 3: MALDI MS data processing and analysis.
    Keywords:  MALDI mass spectrometry imaging; brain; drug response; efavirenz; lipids
    DOI:  https://doi.org/10.1002/cpz1.70108
  32. Int Urogynecol J. 2025 Feb 24.
    Urinary Metabolomic Profile is Minimally Impacted by Common Storage Conditions and Additives.
    Kelly C Weldon, Morgan Panitchpakdi, Andrés Mauricio Caraballo-Rodríguez, Alan J Wolfe, Pieter C Dorrestein, Linda Brubaker, Lindsey A Burnett.
       BACKGROUND: Metabolomics reflects the molecular communications within biological systems. Urine is a noninvasive biofluid, rich in metabolites that serve as potential biomarkers for human health and disease. The impact of storage conditions and DNA stabilizers for urine samples in metabolomic studies remain unclear.
    OBJECTIVE: To evaluate the impact of common storage conditions and the presence of a DNA stabilizer, AssayAssure® (Thermo Scientific), on the metabolite content of voided human urine.
    METHODS: We assessed the urinary metabolite composition under different storage conditions and with the addition of AssayAssure® to determine its effect on metabolomic analysis.
    RESULTS: Urinary metabolite composition remained consistent across different storage conditions. However, the addition of AssayAssure® significantly altered the metabolic profile due to adduct formation. Despite these alterations, the identification of parent metabolites was not compromised, and biological differences were still distinguishable.
    CONCLUSION: These findings suggest that urine biobanked under the tested storage conditions is suitable for metabolomic analysis. The addition of AssayAssure® does not hinder the detection of parent metabolites, although it may affect the overall metabolic profile.
    Keywords:  Urinary metabolites; Urinary metabolome; Urinary metabolomics; Urine storage and preservation
    DOI:  https://doi.org/10.1007/s00192-025-06069-2
  33. Pharmaceutics. 2025 Feb 06. pii: 200. [Epub ahead of print]17(2):
    A Novel LC-MS/MS Method for the Measurement of Elexacaftor, Tezacaftor and Ivacaftor in Plasma, Dried Plasma Spot (DPS) and Whole Blood in Volumetric Absorptive Microsampling (VAMS) Devices.
    Federica Pigliasco, Alessia Cafaro, Sebastiano Barco, Federico Cresta, Rosaria Casciaro, Nicoletta Pedemonte, Francesca Mattioli, Carlo Castellani, Giuliana Cangemi.
      Background: The combination of ivacaftor, tezacaftor and elexacaftor (ETI) is approved for patients with cystic fibrosis (CF) aged two years and older and at least one F508del mutation in the CFTR gene. Variability in ETI treatment response has been repeatedly reported, and its reasons are unclear and understudied. Objectives: We present a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and simultaneous quantification of ETI in plasma, dried plasma spots (DPS), and whole blood volumetric absorptive microsampling (VAMS). Methods: The method utilizes a rapid extraction protocol with 200 μL methanol after the addition of deuterated internal standards. Chromatographic separation was achieved using a reversed-phase Hypersil Gold aQ column (Thermo Fisher Scientific). The method was validated according to ICH (International Council on Harmonisation) guidelines M10 for bioanalytical method validation, demonstrating linearity in the concentration range 0.020-12.000 µg/mL. It was also proved accurate and reproducible with no matrix effect. This method was applied to anonymized samples from patients undergoing ETI treatment: eight plasma and DPS and five VAMS samples were analyzed. Results: ETI concentrations measured in plasma and DPS were interchangeable, whereas ETI concentrations in VAMS were lower than in plasma, as expected for molecules with high plasma protein binding (99%). A correction factor based on the hematocrit value was used to calculate the equivalent plasma concentration from VAMS concentrations. Conclusions: This method is suitable for pharmacokinetic (PK) studies and could facilitate the centralization of samples to specialized laboratories, supporting multicenter studies.
    Keywords:  CFTR modulators; cystic fibrosis; liquid chromatograph–tandem mass spectrometry; microsampling; pediatric pharmacology
    DOI:  https://doi.org/10.3390/pharmaceutics17020200
  34. Anal Chem. 2025 Feb 25.
    Exploring Single-Probe Single-Cell Mass Spectrometry: Current Trends and Future Directions.
    Deepti Bhusal, Shakya Wije Munige, Zongkai Peng, Zhibo Yang.
      The Single-probe single-cell mass spectrometry (SCMS) is an innovative analytical technique designed for metabolomic profiling, offering a miniaturized, multifunctional device capable of direct coupling to mass spectrometers. It is an ambient technique leveraging microscale sampling and nanoelectrospray ionization (nanoESI), enabling the analysis of cells in their native environments without the need for extensive sample preparation. Due to its miniaturized design and versatility, this device allows for applications in diverse research areas, including single-cell metabolomics, quantification of target molecules in single cell, MS imaging (MSI) of tissue sections, and investigation of extracellular molecules in live single spheroids. This review explores recent advancements in Single-probe-based techniques and their applications, emphasizing their potential utility in advancing MS methodologies in microscale bioanalysis.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06824
  35. J Xenobiot. 2025 Feb 02. pii: 23. [Epub ahead of print]15(1):
    New Standardized Procedure to Extract Glyphosate and Aminomethylphosphonic Acid from Different Matrices: A Kit for HPLC-UV Detection.
    Francesco Chiara, Sarah Allegra, Elisa Arrigo, Daniela Di Grazia, Francesco Maximillian Anthony Shelton Agar, Raluca Elena Abalai, Sara Gilardi, Silvia De Francia, Daniele Mancardi.
       BACKGROUND: Glyphosate has been extensively used as herbicide since the early 1970s. The daily exposure limit is set at 0.3 mg/kg bw/d in Europe and 1.75 mg/kg bw/d in the USA. Among its derivatives, aminomethylphosphonic acid is the most stable and abundant. Understanding their biological effects then requires reliable methods for quantification in biological samples.
    METHODS: We developed and validated a fast, low-cost, and reliable chromatographic method for determining glyphosate and aminomethylphosphonic acid concentrations. The validation included following parameters: specificity, selectivity, matrix effect, accuracy, precision, calibration performance, limit of quantification, recovery, and stability. Sample extraction employed an anion exchange resin with elution using hydrochloric acid 50.0 mmol/L. For HPLC analysis, analytes were derivatized, separated on a C18 column with a mobile phase of phosphate buffer (0.20 mol/L, pH 3.0) and acetonitrile (85:15), and detected at 240 nm.
    RESULTS: The method demonstrated high reliability and reproducibility across various matrices. Its performance met all validation criteria, confirming its suitability for quantifying glyphosate and aminomethylphosphonic acid in different biological and experimental setups.
    CONCLUSIONS: This method can offer a practical resource for applications in experimental research, medical diagnostics, quality control, and food safety.
    Keywords:  AMPA; HPLC-UV; biological matrix; glyphosate; liquid chromatography
    DOI:  https://doi.org/10.3390/jox15010023
  36. Environ Sci Technol. 2025 Feb 27.
    IodoFinder: Machine Learning-Guided Recognition of Iodinated Chemicals in Nontargeted LC-MS/MS Analysis.
    Tingting Zhao, Qiming Shen, Xing-Fang Li, Tao Huan.
      Iodinated disinfection byproducts (I-DBPs) pose significant health concerns due to their high toxicity. Current approaches to recognize unknown I-DBPs in mass spectrometry (MS) analysis rely on negative ionization mode, in which the characteristic I- fragment can be observed in tandem mass spectra (MS/MS). Still, many I-DBPs ionize exclusively in positive ionization mode, where the I- fragment is absent. To address this gap, this work developed a machine learning-based strategy to recognize iodinated compounds (I-compounds) from their MS/MS in both electrospray positive (ESI+) and negative ionization (ESI-) modes. Investigating over 6000 MS/MS spectra of 381 I-compounds, we first identified five characteristic I-containing neutral losses and one diagnostic I- fragment in ESI+ and ESI- modes, respectively. We then trained Random Forest models and integrated them into IodoFinder, a Python program, to streamline the recognition of I-compounds from raw LC-MS data. IodoFinder accurately recognized over 96% of the 161 I-compound standards in both ionization modes. In its application to DBP mixtures, IodoFinder discovered 19 I-DBPs with annotated structures and an additional 17 with assigned formulas, including 12 novel and 3 confirmed I-DBPs. We envision that IodoFinder will advance the identification of both known and unknown I-compounds in exposome studies.
    Keywords:  exposome; fragmentation pattern; high-resolution mass spectrometry; iodinated compound; iodinated disinfection byproducts; machine learning; nontargeted analysis
    DOI:  https://doi.org/10.1021/acs.est.4c12698
  37. Anal Chem. 2025 Feb 25.
    Ultrasensitive Determination of Amino Acids in Single Cells by Chemical Isotope Labeling with Liquid Chromatography Mass Spectrometry Analysis.
    Li Ge, Chuanling Jiang, Chengjie Ma, Chun-Yue Han, Yi Gong, Lili Zhu, Qi Liu, Fei-Long Liu.
      Amino acids play multiple critical roles in the regulation of various metabolic pathways and physiological processes in living organisms. Mass spectrometry (MS) has become the most pioneering platform for amino acid analysis. However, the simultaneous and sensitive determination of amino acids is still challenging because of their structural similarity and broad ranges of concentrations. To this end, a pair of isotope labeling reagents, d0/d3-2-((diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl) methanone (DMPI/d3-DMPI), were applied to label amino acid metabolites. The diazo groups on the pair of isotopomers (DMPI/d3-DMPI) can specifically react with the carboxyl groups on the amino acids. The results showed that the retention on reversed-phase column were enhanced and the detection sensitivities of 19 amino acids were increased benefiting from DMPI labeling strategy that transfers the hydrophobic indole heterocycle group of DMPI to the hydrophilic compounds of amino acids. The obtained limits of detection (LODs) of amino acids were in the range of 0.002-0.082 fmol. With this established method, we achieved the sensitive detection of amino acids in a single HUVE cell. Meanwhile, we found that the contents of amino acids in the serum of premature neonates were higher compared to normal neonates. Overall, this developed method provides great support of detection tool for the clinical metabolomic study of amino acids and the investigation of dynamic changes of amino acid metabolism in single cells.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06441
  38. JACS Au. 2025 Feb 24. 5(2): 653-665
    A Systematic Approach to Discover New Natural Product Scaffolds Using Database-Derived Relative Mass Spectral Defects and Molecular Networking.
    Hyo Moon Cho, Eleonora Boccia, Rahim Rajwani, Robert D O'Connor, Helena Boshoff, Clifton Barry, Giuseppe Bifulco, Carole A Bewley.
      Rapid advances in mass spectrometry (MS) data analysis have accelerated the identification of natural products from complex mixtures such as natural product extracts. However, limitations in MS data in metabolite libraries and dereplication strategies are still lacking for assigning structures to known compounds and searching for unidentified compounds. To overcome these limitations, we present an approach that combines molecular networking with MS database-derived mass defect analysis to preferentially discover new compounds with high structural novelty in the initial stage of a discovery workflow. Specifically, unknown metabolites or clusters generated from molecular networking are assigned to a compound class based on their relative mass defects (RMDs) calculated using open-source databases. If ancillary data such as ultraviolet and MS/MS spectra of the unknown clusters are incongruent with the RMD-assigned compound class, metabolites are considered to have a new skeleton that exhibits a large difference in RMD value due to structural changes. Here, we applied this RMD-assisted method to a desert-derived bacterial strain library and validated it through the discovery of brasiliencin A (1), a new 18-membered macrolide from Nocardia brasiliensis. A putative biosynthetic pathway of brasiliencin A was proposed through whole-genome sequence analysis, and an additional 29 analogs were detected using absolute mass defect filtering (AMDF) based on plausible biosynthetic products. This led to the isolation of three additional macrolides, brasiliencins B-D (2-4). The structures of the brasiliencins (1-4) were fully elucidated through spectroscopic data analysis and quantum chemical calculations including ROE distance and 13C NMR chemical shift calculations, and experimental and theoretical electronic circular dichroism (ECD). Brasiliencin A showed strong activity against Mycobacterium smegmatis and Streptococcus australis (MIC = 31.3 nM and 7.81 μM, respectively) compared to brasiliencin B (MIC = 1000 nM and 62.5 μM, respectively) that differs at a single stereocenter.
    DOI:  https://doi.org/10.1021/jacsau.4c00889
  39. Anal Bioanal Chem. 2025 Feb 26.
    Two-dimensional isotopic shifts for steroid isomer delineation with high-resolution cyclic ion mobility separations.
    Noah D Roberts, Gabriella Sprague, Gabe Nagy.
      Recently, the use of mass distribution-based isotopic shifts in high-resolution ion mobility spectrometry-mass spectrometry-based separations have enabled isomer delineation by measuring the relative arrival times of their heavy and light isotopologues. However, all previous efforts to induce such shifts have focused solely on the introduction of one type of isotopic substitution for a given molecule or isomer set. Herein, for the first time, we present a two-dimensional isotopic labeling strategy where two unique derivatizations are performed on various steroid isomer molecules to induce two distinct isotopic shifts and thus simultaneously measure them in a single ion mobility separations experiment. Derivatization strategies were chosen to target two specific functional groups in these steroids (i.e., hydroxyl and carbonyl), and heavy-labeled versions of the derivatizing reagents were used to induce isotopic shifts at each of these positions. We found that isotopic shifts were orthogonal to one another, diagnostic for certain steroid isomers, and that the simultaneous analysis of two different isotopic shifts was necessary for complete characterization of each steroid isomer set. We envision this multidimensional isotopic shift strategy as a new method for delineating amongst isomeric molecules, especially those with several different functional groups causing their isomerism.
    Keywords:  Cyclic ion mobility spectrometry; High-resolution ion mobility; Isotopic shifts; Mass spectrometry; Steroids
    DOI:  https://doi.org/10.1007/s00216-025-05806-2
  40. Molecules. 2025 Feb 07. pii: 779. [Epub ahead of print]30(4):
    Utilizing High-Resolution Mass Spectrometry Data Mining Strategy in R Programming Language for Rapid Annotation of Absorbed Prototypes and Metabolites of Gypenosides.
    Xiaoshan Li, Qianru Zhang, Yuqin Li, Lin Qin, Di Wu, Daopeng Tan, Jian Xie, Jiajia Wu, Qingping Yang, Yanliu Lu, Yongxia Zhao, Qingjie Fan, Xingdong Wu, Yuqi He.
      The rapid and accurate annotation of the complex compounds and metabolites in natural products remains a significant challenge. In this study, we developed an integrated strategy to efficiently and accurately profile both the prototypes and metabolites of natural products in vivo. And this was achieved by establishing a gypenosides constituent database and utilizing R programming language to combine sample selection, virtual metabolite database construction, polygon mass defect filtering, and Kendrick mass defect filtering. In addition, the annotation strategy was successfully applied to identify the prototypes and metabolites of gypenosides in mice serum. As a case study, gypenoside LXXV was used to validate the feasibility of this approach. The results demonstrated 36 prototypes and 108 metabolites were annotated from the serum by the established annotation strategy. The prototype and eight metabolites of gypenoside LXXV were further confirmed, indicating that the proposed strategy is available. This study provides a novel approach for the rapid and accurate identification of prototypes and metabolites of natural products and offers new insights into the metabolic processes of gypenosides in vivo.
    Keywords:  R programming language; gypenosides; mass defect; metabolite annotation; virtual metabolite library
    DOI:  https://doi.org/10.3390/molecules30040779
  41. J Pharm Biomed Anal. 2025 Feb 11. pii: S0731-7085(25)00082-2. [Epub ahead of print]259 116741
    Molecular networking: An efficient tool for discovering and identifying natural products.
    Yongjian Wang, Yadan Wang, Zhongmou Zhang, Kailing Xu, Qiufang Fang, Xianfu Wu, Shuangcheng Ma.
      Natural products (NPs), play a crucial role in drug development. However, the discovery of NPs is accidental, and conventional identification methods lack accuracy. To overcome these challenges, an increasing number of researchers are directing their attention to Molecular networking (MN). MN based on secondary mass spectrometry has become an important tool for the separation, purification and structural identification of NPs. However, most new tools are not well known. This review started with the most basic MN tool and explains it from the principle, workflow, and application. Then introduce the principles and workflows of the remaining eight new types of MN tools. The reliability of various MNs is mainly verified based on the application of phytochemistry and metabolomics. Subsequently, the principles and applications of 12 structural annotation tools are introduced. For the first time, the scope of 9 kinds of MN tools is compared horizontally, and 12 kinds of structured annotation tools are classified from the type of compound structure suitable for identification. The advantages and disadvantages of various tools are summarized, and make suggestions for future application directions and the development of computing tools in this review. MN tools are expected to enhance the efficiency of the discovery and identification in NPs.
    Keywords:  Molecular networking; Molecular networking with special functions; Natural products; Secondary mass spectrometry; Structural annotation tools
    DOI:  https://doi.org/10.1016/j.jpba.2025.116741
  42. Anal Bioanal Chem. 2025 Feb 22.
    GlycoDash: automated, visually assisted curation of glycoproteomics datasets for large sample numbers.
    Tamas Pongracz, Steinar Gijze, Agnes L Hipgrave Ederveen, Rico J E Derks, David Falck.
      The challenge of robust and automated glycopeptide quantitation from liquid chromatography-mass spectrometry (LC-MS) data has yet to be adequately addressed by commercial software. Recently, open-source tools like Skyline and LaCyTools have advanced the field of label-free MS1 level quantitation. Yet, important steps late in the data processing workflow remain manual. Because manual data curation is time-consuming and error-prone, it presents a bottleneck, especially in an era of emerging high-throughput methodologies and increasingly complex analyses such as antigen-specific antibody glycosylation. We addressed this gap by developing GlycoDash, an R Shiny-based interactive web application designed to democratize label-free high-throughput glycoproteomics data analysis. The software comes in at a stage where analytes have been identified and quantified, but whole measurement and individual analyte signals of insufficient quality for quantitation remain and reduce the quality of the overall dataset. GlycoDash focuses on these challenges by incorporating several options for measurement and metadata linking, spectral and analyte curation, normalization, and repeatability assessment, and additionally includes glycosylation trait calculation, data visualization, and reporting capabilities that adhere to FAIR principles. The performance and versatility of GlycoDash were demonstrated across antibody glycoproteomics data of increasing complexity, ranging from relatively simple monoclonal antibody glycosylation analysis to a clinical cohort with over a thousand measurements. In a matter of hours, these large, diverse, and complex datasets were curated and explored. High-quality datasets with integrated metadata ready for final analysis and visualization were obtained. Critical aspects of the curation strategy underlying GlycoDash are discussed. GlycoDash effectively automates and streamlines the curation of glycopeptide quantitation data, addressing a critical need for high-throughput glycoproteomics data analysis. Its robust performance across diverse datasets and its comprehensive feature toolbox significantly enhance both research and clinical applications in glycoproteomics.
    Keywords:  Antibody glycosylation; Data curation; Glycomics software; Glycoproteomics; High-throughput glycomics; LC–MS
    DOI:  https://doi.org/10.1007/s00216-025-05794-3
  43. J Proteome Res. 2025 Feb 28.
    pyOpenMS-viz: Streamlining Mass Spectrometry Data Visualization with pandas.
    Justin Cyril Sing, Joshua Charkow, Axel Walter, Mingxuan Gao, Tom David Müller, Wout Bittremieux, Timo Sachsenberg, Hannes Luc Röst.
      Mass spectrometry data visualization is essential for a wide range of applications, such as validation of workflows and results, benchmarking new algorithms, and creating comprehensive quality control reports. Python offers a popular and powerful framework for analyzing and visualizing multidimensional data; however, generating commonly used mass spectrometry plots in Python can be cumbersome. Here we present pyOpenMS-viz, a versatile, unified framework for generating mass spectrometry plots. pyOpenMS-viz directly extends pandas DataFrame plotting for generating figures in a single line of code. This implementation enables easy integration across various Python-based mass spectrometry tools that already use pandas DataFrames to store MS data. pyOpenMS-viz is open-source under a BSD 3-Clause license and freely available at https://github.com/OpenMS/pyopenms_viz.
    Keywords:  mass-spectrometry; python; quality control; validation; visualization
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00873
  44. Biologicals. 2025 Feb 24. pii: S1045-1056(25)00013-2. [Epub ahead of print]90 101822
    Ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) for simultaneous estimation of residual glyphosate and its metabolite (amino methyl phosphonic acid - AMPA) in various vaccines.
    Bharat Shinde, Dadasaheb Patil, Nitin Kadam, Manish Gautam, Kaushik Banerjee, Sunil Gairola, Pooja Doshi.
      There is growing interest of monitoring of glyphosate (GLYP) and its active metabolite, aminomethylphosphonic acid (AMPA) in pharmaceuticals globally. Vaccines represents an important class of pharmaceuticals for human and veterinary use. In this work, a robust, sensitive and direct ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) based method was developed and validated. This method enables simultaneous detection and quantification of GLYP and AMPA using a simple liquid-liquid extraction technique in complex vaccines. In absence of these residuals, the method was validated using spiked standards of GLYP and AMPA in the selected vaccines. The method demonstrated suitable linearity with r2 > 0.997 over the wide concentration range of 2-50 ng/mL for GLYP and 2-100 ng/mL for AMPA respectively. LOD and LOQ of 0.5 ng/mL and 2 ng/mL for GLYP and AMPA was observed. The method showed precision (RSD of 14 %) and accuracy (83-108 %) in selected vaccines including diphtheria-tetanus-whole cell pertussis-hepatitis B and haemophilus influenzae type B conjugate, diphtheria-tetanus-whole cell pertussis-hepatitis B-haemophilus influenzae type B conjugate and inactivated polio virus, measles-mumps-rubella and pneumococcal polysaccharide conjugate vaccines. The study supports the suitability of the method for simultaneous monitoring of GLYP and AMPA in vaccine formulations.
    Keywords:  Aminomethylphosphonic acid; Contaminants; Glyphosate; UPLC-MS/MS; Vaccine; Validation
    DOI:  https://doi.org/10.1016/j.biologicals.2025.101822
  45. Food Res Int. 2025 Mar;pii: S0963-9969(25)00255-8. [Epub ahead of print]204 115918
    Analysis of germination and vigor of cashew seeds for germplasm conservation by NMR and uPLC-qTOF-MS/MS based metabolomics.
    Elenilson Godoy Alves Filho, João Ravelly Alves de Queirós, Oriel Herrera Bonilla, Hilton César Rodrigues Magalhães, Edy Sousa de Brito, Paulo Riceli Vasconcelos Ribeiro, Kirley Marques Canuto, Lorena Mara Alexandre E Silva, Ana Cecília Ribeiro de Castro.
      The effective preservation of cashew germplasm faces significant challenges due to the loss of germination vigor over medium and long periods. Therefore, this study aimed to assess the biometric and chemical parameters that determine the germination and vigor of cashew seed. Seeds from seven cashew genotypes stored under refrigeration (18 ± 2 °C) were evaluated for biometric characteristics and post-emergence development analyzed by NMR, UPLC-QTOF-MSE and GC-MS methods for profiling primary and secondary metabolites along with fatty acids. The evaluated materials revealed significant variability in emergence percentage (0 % to 91.43 %), exhibiting a negative correlation with biometric characteristics. The emergence speed index (ESI) and emergence speed (ES) ranged from 1.20 to 3.61 and 16.58 to 22.84 days, respectively. The oleic acid is the major fatty acid (60 %) and the correlation of the fatty acid content with emergency remain unclear. The NMR and UPLC-QTOF-MSE analysis highlighted the compositional variability among the genotypes. In this regard, NMR and statistical modeling shows positive correlation which seeds with higher stachyose and acetic acid and lower sucrose contents exhibited greater germination. Additionally, seeds with enhanced germination parameters positively correlated with greater phenolic lipids content, such as anacardic acids. Therefore, this study indicates that seeds with reduced biometric parameters that presented increased content of stachyose, acetic acid, and anacardic acids were correlated higher emergence capacity making those seeds more suitable to cashew germplasm cryopreservation banks.
    Keywords:  Anacardium occidentale; Germplasm conservation banks; NMR; NMR based metabolomics; Seed storage
    DOI:  https://doi.org/10.1016/j.foodres.2025.115918
  46. Biomolecules. 2025 Feb 08. pii: 246. [Epub ahead of print]15(2):
    Optimized Extraction Method for Neutral Cannabinoids Quantification Using UHPLC-HRMS/MS.
    João Victor Meirelles, Débora Cristina Diniz Estevam, Vanessa Farelo Dos Santos, Henrique Marcelo Gualberto Pereira, Tatiana D Saint Pierre, Valdir F Veiga-Junior, Monica Costa Padilha.
      The Cannabis market is experiencing steady global growth. Cannabis herbal extracts (CHE) are interesting and sought-after products for many clinical conditions. The medical potential of these formulations is mainly attributed to neutral cannabinoids, such as cannabidiol (CBD), tetrahydrocannabinol (THC), and cannabinol (CBN), and their non-standardized content poses a significant fragility in these pharmaceutical inputs. High-resolution mass spectrometry portrays a powerful alternative to their accurate monitoring; however, further analytical steps need to be critically optimized to keep up with instrumental performance. In this study, Full Factorial and Box-Behnken designs were employed to achieve a multivariate optimization of CBD, THC, and CBN extraction from human and veterinary commercial CHE using a minimum methanol/hexane 9:1 volume and short operational times. A predictive model was also constructed using the Response Surface Methodology and its accuracy was validated. Agitation and sonication times were identified as the most significant operational extraction parameters, followed by the extraction mixture volume. The final setup of the optimized method represented a significantly faster and cheaper protocol than those in the literature. The selected neutral cannabinoids quantification was conducted using ultra high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) with a precision of <15%, accuracy of 69-98%, sensitivity of 23-39 ng kg-1, and linearity regarding pharmaceutical requirements. State-of-the-art levels of analytical sensitivity and specificity were achieved in the target quantification due to high-resolution mass spectrometry. The developed method demonstrated reliability and feasibility for routine analytical applications. As a proof-of-concept, it enabled the efficient processing of 16 samples of commercial CHE within a three-hour timeframe, showcasing its practicality and reproducibility, and highlighting its potential for broader adoption in similar scenarios for both human and veterinary medicines.
    Keywords:  Box–Behnken design; Cannabis herbal extracts; Full Factorial Design; UHPLC-HRMS/MS; cannabinoids
    DOI:  https://doi.org/10.3390/biom15020246
  47. J Am Soc Mass Spectrom. 2025 Feb 25.
    To Impute or Not To Impute in Untargeted Metabolomics─That is the Compositional Question.
    Dennis D Krutkin, Sydney Thomas, Simone Zuffa, Prajit Rajkumar, Rob Knight, Pieter C Dorrestein, Scott T Kelley.
      Untargeted metabolomics often produce large datasets with missing values. These missing values are derived from biological or technical factors and can undermine statistical analyses and lead to biased biological interpretations. Imputation methods, such as k-Nearest Neighbors (kNN) and Random Forest (RF) regression, are commonly used, but their effects vary depending on the type of missing data, e.g., Missing Completely At Random (MCAR) and Missing Not At Random (MNAR). Here, we determined the impacts of degree and type of missing data on the accuracy of kNN and RF imputation using two datasets: a targeted metabolomic dataset with spiked-in standards and an untargeted metabolomic dataset. We also assessed the effect of compositional data approaches (CoDA), such as the centered log-ratio (CLR) transform, on data interpretation since these methods are increasingly being used in metabolomics. Overall, we found that kNN and RF performed more accurately when the proportion of missing data across samples for a metabolic feature was low. However, these imputations could not handle MNAR data and generated wildly inflated or imputed values where none should exist. Furthermore, we show that the proportion of missing values had a strong impact on the accuracy of imputation, which affected the interpretation of the results. Our results suggest imputation should be used with extreme caution even with modest levels of missing data and especially when the type of missingness is unknown.
    DOI:  https://doi.org/10.1021/jasms.4c00434
  48. Anal Chem. 2025 Feb 25.
    Network Flow Methods for NMR-Based Compound Identification.
    Leonhard Lücken, Nico Mitschke, Thorsten Dittmar, Bernd Blasius.
      In this work, we introduce a novel method for compound identification in mixtures based on nuclear magnetic resonance spectra. Contrary to many other methods, our approach can be used without peak-picking the mixture spectrum and simultaneously optimizes the fit of all individual compound spectra in a given library. At the core of the method, a minimum cost flow problem is solved on a network consisting of nodes that represent spectral peaks of the library compounds and the mixture. We show that our approach can outperform other popular algorithms by applying it to a standard compound identification task for 2D 1H,13C HSQC spectra of artificial mixtures and a natural sample using a library of 501 compounds. Moreover, our method retrieves individual compound concentrations with at least semiquantitative accuracy for artificial mixtures with up to 34 compounds. A software implementation of the minimum cost flow method is available on GitHub (https://github.com/GeoMetabolomics-ICBM/mcfNMR).
    DOI:  https://doi.org/10.1021/acs.analchem.4c01652
  49. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Feb 18. pii: S1570-0232(25)00078-9. [Epub ahead of print]1255 124526
    Imatinib and norimatinib therapeutic monitoring using dried blood spots: Analytical and clinical validation, and performance comparison of volumetric collection devices.
    Marco Orleni, Sara Gagno, Eleonora Cecchin, Marcella Montico, Angela Buonadonna, Arianna Fumagalli, Michela Guardascione, Fabio Puglisi, Giuseppe Toffoli, Bianca Posocco, Erika Cecchin.
      Therapeutic drug monitoring during imatinib treatment is recommended to optimize patient clinical outcomes. This study aimed to develop a novel LC-MS/MS method to quantitate imatinib and its active metabolite N-desmethyl-imatinib, in volumetric dried blood spots (DBS) using the HemaXis DB10 and Capitainer B devices. Chromatographic separation was achieved using an XTerra MS C18 column and detection occurred with a SCIEX 4000QTrap tandem mass spectrometer using electrospray positive-mode ionization. Analytical validation was successfully performed adhering to the latest guidelines. The assay was linear over the range 240-6000 ng/mL for imatinib and 48-1200 ng/mL for its metabolite, accurate (89 %-113 %) and precise (≤17 % imprecision) across a hematocrit range of 22-55 % for both devices. Recovery ranged from 84 % to 92 %, with no influence of matrix components. Stability was confirmed after at least 43 days in desiccator conditions (20 °C, ≤35 % humidity), and in conditions that mimed home-sampling. Clinical validation, conducted on 52 paired DBS and plasma samples from 28 patients, revealed that the DBS-to-plasma ratio can be used to convert DBS measurements into plasma concentrations. Bland-Altman and Passing-Bablok analyses indicated strong agreement between the estimated and actual plasma concentrations for both imatinib and its metabolite across both devices. The conversion method was further tested on an additional set of 25 to 31 samples, with 80 to 97 % of the samples falling within ±20 % difference. This study proved that DBS collected using either HemaXis DB10 or Capitainer B devices can be reliably implemented as an alternative to plasma for therapeutic drug monitoring during imatinib therapy.
    Keywords:  Capitainer B; Dried blood spot; HemaXis DB10; Imatinib; LC-MS/MS; Pharmacokinetics; Therapeutic drug monitoring
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124526
  50. Biomolecules. 2025 Feb 07. pii: 243. [Epub ahead of print]15(2):
    Microbiota-Gut-Brain Axis: Mass-Spectrometry-Based Metabolomics in the Study of Microbiome Mediators-Stress Relationship.
    Nicolò Interino, Rosalba Vitagliano, Federica D'Amico, Raffaele Lodi, Emanuele Porru, Silvia Turroni, Jessica Fiori.
      The microbiota-gut-brain axis is a complex bidirectional communication system that involves multiple interactions between intestinal functions and the emotional and cognitive centers of the brain. These interactions are mediated by molecules (metabolites) produced in both areas, which are considered mediators. To shed light on this complex mechanism, which is still largely unknown, a reliable characterization of the mediators is essential. Here, we review the most studied metabolites in the microbiota-gut-brain axis, the metabolic pathways in which they are involved, and their functions. This review focuses mainly on the use of mass spectrometry for their determination, reporting on the latest analytical methods, their limitations, and future perspectives. The analytical strategy for the qualitative-quantitative characterization of mediators must be reliable in order to elucidate the molecular mechanisms underlying the influence of the above-mentioned axis on stress resilience or vulnerability.
    Keywords:  mass spectrometry; mediators; metabolomics; microbiota–gut–brain axis; stress
    DOI:  https://doi.org/10.3390/biom15020243
  51. Toxins (Basel). 2025 Jan 23. pii: 52. [Epub ahead of print]17(2):
    Contamination Status and Health Risk Assessment of 73 Mycotoxins in Four Edible and Medicinal Plants Using an Optimized QuEChERS Pretreatment Coupled with LC-MS/MS.
    Xiaojing Huang, Rui Feng, Qing Hu, Xiuhong Mao, Heng Zhou.
      The current status of multi-mycotoxin contamination in edible and medicinal plants demands urgent development of high-throughput analytical methods for mycotoxin detection. In this study, a reliable and sensitive method for the simultaneous analysis of 73 mycotoxins was established and successfully applied to detect mycotoxins in 260 samples of four dual-purpose plants (lotus seed, coix seed, licorice root, and dried tangerine peel). Sample preparation involved optimized QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction combined with liquid-liquid extraction purification, and an enhanced ion pair library was established to reduce matrix interference and improve the method's universality. Method validation demonstrated recovery rates ranging from 61.6% to 118.6% for all compounds, with relative standard deviations (RSDs) below 15%. The limits of detection (LODs) and quantification (LOQs) ranged from 0.25-12.25 μg/kg and 0.5-25 μg/kg, respectively. Based on the contamination analysis and health risk assessment using Margin of Exposure (MOE) and Hazard Index (HI) methods, we found that multi-mycotoxin contamination is highly prevalent in edible and medicinal plants, with different components being susceptible to invasion by distinct fungal genera. Seed-type plants showed high susceptibility to Aspergillus (53.3%) and Fusarium (22.2%) contamination, with MOE values below 10,000 for aflatoxins indicating potential health risks. Physical state and good storage conditions significantly influenced contamination levels, with fragmented samples showing substantially higher mycotoxin levels. Additionally, mycotoxins with associated biosynthetic metabolic pathways were frequently detected simultaneously in highly contaminated samples. Based on these findings, we recommend implementing strict moisture control during storage, maintaining intact product form where possible, and establishing comprehensive supplier qualification systems. This study provides valuable reference for monitoring mycotoxin contamination in similar plants.
    Keywords:  QuEChERS; UHPLC-MS/MS; contamination status; edible and medicinal plants; health risk assessment; mycotoxins
    DOI:  https://doi.org/10.3390/toxins17020052
  52. J Pharm Biomed Anal. 2025 Feb 19. pii: S0731-7085(25)00089-5. [Epub ahead of print]259 116748
    Analytical validation and pilot clinical application of a UPLC-MS/MS method for determining intracellular mycophenolic acid and metabolites in kidney transplant recipients.
    Xiaomei Chen, Xinhua Dai, Huan Xu, Chunxia Chen, Xueqaio Wang, Yuangao Zou, Hanjing Liu, Yunying Shi, Yi Li, Yangjuan Bai.
      There is no consensus on the strategy for therapeutic drug monitoring of the immunosuppressive drug mycophenolic acid (MPA) in organ transplant recipients. The present study proposes the utilization of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for determining the concentrations of MPA and its metabolites: 7-O-mycophenolic acid glucuronide (MPAG) and acyl mycophenolic acid glucoside (AcMPAG) in peripheral blood mononuclear cells (PBMCs). We aimed to assess the potential application of monitoring MPA and its metabolite concentrations in PBMCs in the infection after transplantation in Chinese kidney transplant recipients (KTRs). The UPLC-MS/MS method we developed demonstrated good linearity in the quantitative ranges of 0.05-50.00 ng/mL for MPA, 0.50-50.00 ng/mL for MPAG, and 0.10-20.00 ng/mL for AcMPAG. AcMPAG in PBMCs was unstable, degrading significantly after 48 h of storage at -80°C or after 3 freeze-thaw cycles. MPA and MPAG concentrations in KTRs' PBMCs exhibited high inter-individual variability, and the MPA concentration in PBMCs was poorly correlated with that in plasma (rs = 0.206, p = 0.117). Compared with the stable group, the infected group had significantly higher MPA concentration in PBMCs at 2 and 4 h post-dosing and in plasma at 4 h post-dosing (p < 0.05). The receiver operating characteristic (ROC) analysis for post-transplantation infection revealed that PBMCs MPA-C4 and PBMCs-MPA-C2 possessed much better diagnostic efficiency than Plasma-MPA-C4. This method is easy-to-use and reliable, making it a promising clinical quantitative tool for MPA, MPAG, and AcMPAG in PBMCs. PBMC-MPA monitoring may be a potential biomarker for infection monitoring for KTRs.
    Keywords:  7-O-mycophenolic acid glucuronide; Acyl-mycophenolic acid glucoside; Infection; Mycophenolic acid; Peripheral blood mononuclear cell; Ultra-performance liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.jpba.2025.116748
  53. BMC Chem. 2025 Feb 22. 19(1): 50
    Development of a novel UHPLC-MS/MS method for quantitative analysis of pirtobrutinib in rat plasma: application to pharmacokinetic study.
    Meijuan Zhang, Jingxuan Wu, Jiangshuo Li, Hang Yin, Mengyu Hou, Ruihua Dong.
      Bruton's tyrosine kinase (BTK) inhibitors play a critical role in the treatment of mantle cell lymphoma (MCL). pirtobrutinib, a new, highly selective, non-covalent BTK inhibitor, was approved by the FDA for the treatment of MCL, chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL). In this study, we established a robust and reliable method for the quantitation of pirtobrutinib in rat plasma using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Acetonitrile and 0.1% formic acid served as the mobile phase, with zanubrutinib as the internal standard (IS). Detection ion transitions were m/z 480.12→294.05 for pirtobrutinib and m/z 472.20→ 289.96 for Zanubrutinib. The intra-day and inter-day relative standard deviation (RSD%) values of pirtobrutinib were less than 9.8% and 10.3%, respectively. Recovery and matrix effects ranged from 95.1 to 101.5% and 91.7-100.4%. In addition, the test sample stability was confirmed under various storage conditions, and this method was successfully applied to a pharmacokinetic study of pirtobrutinib at a dose of 10 mg·kg- 1.
    Keywords:  Pharmacokinetics; Pirtobrutinib; UHPLC-MS/MS; Zanubrutinib
    DOI:  https://doi.org/10.1186/s13065-025-01424-2
  54. Shokuhin Eiseigaku Zasshi. 2025 ;66(1): 12-18
    [Ruhemann's Purple Monitoring by UHPLC/MS/MS for Ninhydrin Test].
    Takashi Kurohara, Chiye Tatebe, Yumiko Fujiwara, Fuyuko Hioki, Shohei Takada, Atsuko Tada, Naoki Sugimoto.
      For amino acids used as food additives, Japan's Specifications and Standards for Food Additives stipulate the ninhydrin test as an identification test. The ninhydrin test is a simple method that involves the visual determination of purple color from the formation of Ruhemann's purple (RP) and does not require special equipment, facilitating its widespread use in society. However, because of this background, objective and molecular selective observation methods for monitoring RP itself as an analyte have not been fully investigated. Therefore, in this study, a UHPLC/MS/MS method was developed to specifically monitor RP and support visual judgment. This method identified RP-derived fragment ions at m/z 170 (ESI (-)) and 133 (ESI (+)), which can be monitored in the multiple reaction monitoring mode and were shown to correlate with the intensity of the purple color. In addition, computational chemistry was applied to scientifically estimate the molecular structures of the fragment ions. In this study, we established a useful analytical method that complements the objectivity of the ninhydrin test. This method is also expected to be utilized for further optimization of test reaction conditions.
    Keywords:  Ruhemann’s purple; UHPLC/MS/MS; amino acid; food additive; ninhydrin
    DOI:  https://doi.org/10.3358/shokueishi.66.12
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