bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–01–19
fifty-one papers selected by
Sofia Costa, Matterworks
  1. Ion Pair Chromatography for Endogenous Metabolite LC-MS Analysis in Tissue Samples Following HGH Resolution Untargeted Acquisition.
  2. Data Treatment for LC-MS Untargeted Analysis.
  3. A Highly Sensitive Triple Quad LC-MS/MS Method Development and Validation for the Determination of Bexicaserin (LP352) in Human Plasma and Urine Matrices.
  4. Quantitative Lipidomics of Biological Samples Using Supercritical Fluid Chromatography Mass Spectrometry.
  5. A sensitive post-column derivatization approach for enhancing hydroxyl metabolites detection.
  6. Untargeted Metabolic Phenotyping by LC-MS.
  7. Quality Control and Validation Issues in LC-MS-Based Metabolomics.
  8. Quantitative Determination of a Series of Oxysterols by an Optimized LC-MS/MS Analysis in Different Tissue Types.
  9. Advanced LC-IMS-MS Protocol for Holistic Metabolite Analysis in Wine and Grape Samples.
  10. Quality Assurance in Metabolomics and Metabolic Profiling.
  11. Spatial metabolomics platform combining mass spectrometry imaging and in-depth chemical characterization with capillary electrophoresis.
  12. Multiclass Steroid Profiling in Short-Finned Pilot Whale Blubber Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
  13. Analysis of Urinary Metanephrines Using Liquid Chromatography Tandem Mass Spectrometry.
  14. OzNOxESI: A Novel Mass Spectrometry Ion Chemistry for Elucidating Lipid Double-Bond Regioisomerism in Complex Mixtures.
  15. UHPLC-TIMS-PASEF®-MS for Lipidomics: From Theory to Practice.
  16. Simultaneous Profiling of Multiple Phosphorylated Metabolites in Typical Biological Matrices via Ion-Pair Reversed-Phase Ultrahigh-Performance Liquid Chromatography and Mass Spectrometry.
  17. Ion mobility spectrometry and ion mobility-mass spectrometry in clinical chemistry.
  18. HILIC-MS/MS Multi-targeted Method for Metabolomics Applications.
  19. Bio- and Chemoinformatic Approaches for Metabolomics Data Analysis.
  20. Enhanced nano-LC-MS for analyzing dansylated oral cancer tissue metabolome dissolved in solvents with high elution strength.
  21. Establishment of a novel large-scale targeted metabolomics method based on NFSWI-DDA mode utilizing HRMS and TQ-MS.
  22. Two-Phase Extraction for Comprehensive Analysis of the Plant Metabolome by NMR.
  23. mpactR: an R adaptation of the metabolomics peak analysis computational tool (MPACT) for use in reproducible data analysis pipelines.
  24. A Disposable Pipette Extraction-UHPLC-MS/MS Method Based on Removal of Phospholipids to Determine Anandamide, 2-Arachidonoylglycerol, Cannabidiol, and Δ9-Tetrahydrocannabidiol in Plasma Samples.
  25. Rat Fecal Metabolomics-Based Analysis.
  26. Hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of oligosaccharides and iridoid glycosides in rat plasma: Pharmacokinetic characterization of previously overlooked oligosaccharides from Radix Rehmanniae.
  27. A reliable LC-MS/MS method for the quantification of natural amino acids in human plasma and its application in clinic.
  28. A Differential Ion Mobility Acoustic Ejection Mass Spectrometer System for Screening Isomerization-Mediating Enzyme Drug Targets.
  29. Determination of osimertinib concentration in rat plasma and lung/brain tissues.
  30. Enhancing lipid identification in LC-HRMS data through machine learning-based retention time prediction.
  31. A novel 3D-printed sample preparation method for benzodiazepine quantification in human serum.
  32. ABCoRT: Retention Time Prediction for Metabolite Identification via Atom-Bond Co-Learning.
  33. A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.
  34. A Novel Approach to the Isolation of Disease Markers: Quantitative Analysis of Amino Acids in Exhaled Breath Condensates from Sleep Apnea Patients with Enhanced Sampling and LC-ESI-MS.
  35. A Novel Liquid Chromatography-Tandem Mass Spectrometry Method to Quantify Tryptophan and Its Major Metabolites in Serum to Support Biomarker Studies in Patients with Cancer Undergoing Immunotherapy.
  36. Development and Validation of a Highly Sensitive Isotope-Coded Equivalent Reporter Ion Assay for the Semi-Quantification of Isocoumarins in Complex Matrices.
  37. Separation of polar and neutral lipids from mammalian cell lines by high-performance thin-layer chromatography.
  38. BiomiX, a user-friendly bioinformatic tool for democratized analysis and integration of multiomics data.
  39. Quality by Design-Steered Chromatographic Separation and Identification of the Geometric Isomers of Capsiate by Reversed-Phase HPLC and LC-MS.
  40. Progress in Tandem Mass Spectrometry Data Analysis for Nucleic Acids.
  41. Exploring the potential of MRM-IDA-EPI method in mass spectrometry for exposomic analysis: a commentary.
  42. Microseparation of Lipophilic and Hydrophilic Metabolites for Single Oocyte Mass Spectrometry Analysis.
  43. Composition-dependent MRM transitions and structure-indicative elution segments (CMTSES)-based LC-MS strategy for disaccharide profiling and isomer differentiation.
  44. iTASO: A Novel Photosensitive Probe for High-Throughput and Selective Submetabolomic Analysis via Flow Injection-Mass Spectrometry.
  45. Beyond the baseline: Quantification of two Phosphatidylethanol (PEth) homologues in Whole Blood by LC-MS/MS and Retrospective Data Analysis from a National Reference Laboratory.
  46. Protein Precipitation by Metal Hydroxides as a Convenient and Alternative Sample Preparation Procedure for Bioanalysis.
  47. A Protocol for GC-MS Profiling of Chiral Secondary Amino Acids.
  48. Feasibility of IR-MALDESI Mass Spectrometry Imaging of PFAS.
  49. Development and Validation of a Robust and Straightforward LC-MS Method for Measuring Taurine in Whole Blood and Plasma of Dogs and Reference Intervals Calculation.
  50. Method Validation for Estimation of Imidacloprid and its Metabolites in Maize and Soil by LCMS-MS.
  51. New Library-Based Methods for Nontargeted Compound Identification by GC-EI-MS.
  1. Methods Mol Biol. 2025 ;2891 165-180
    Ion Pair Chromatography for Endogenous Metabolite LC-MS Analysis in Tissue Samples Following HGH Resolution Untargeted Acquisition.
    Filippos Michopoulos.
      A protocol for the preparation of tissue extracts for the targeted analysis ca. 150 polar metabolites, including those involved in central carbon metabolism, is described, using a reversed phase ion pair U(H)PLC-MS method. Data collection enabled in high-resolution mass spectrometry detection provides highly specific and sensitive acquisition of metabolic intermediates with wide range physicochemical properties and pathway coverage. Technical aspects are discussed for method transfer along with the basic principles of sample sequence setup, data analysis, and validation. At last general comments are given to help the assessment of data quality and system performance.
    Keywords:  Ion pair chromatography; Mass spectrometry; Metabolite profiling; Polar metabolites
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_9
  2. Methods Mol Biol. 2025 ;2891 91-108
    Data Treatment for LC-MS Untargeted Analysis.
    Mar Garcia-Aloy, Johannes Rainer, Pietro Franceschi.
      Liquid Chromatography-Mass Spectrometry (LC-MS) untargeted experiments require complex bioinformatic strategies to extract information from the experimental data. Here we discuss the "data preprocessing," the set of procedures performed on the raw data to produce a data matrix which will be the starting point for the subsequent statistical analysis. Data preprocessing is a crucial step on the path to knowledge extraction, which should be carefully controlled and optimized in order to maximize the output of any untargeted metabolomics investigation.
    Keywords:  Metadata; Missing values; Peak picking; Preprocessing; Quality check; Retention time correction
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_5
  3. Biomed Chromatogr. 2025 Feb;39(2): e6079
    A Highly Sensitive Triple Quad LC-MS/MS Method Development and Validation for the Determination of Bexicaserin (LP352) in Human Plasma and Urine Matrices.
    Raja Reddy Kallem, Maisy Yeager, Rosa Chan, Katharine Fletcher, Katie Neal, Nuggehally R Srinivas.
      Bexicaserin is a highly selective agonist at the 5-HT2c receptor in clinical development for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We report an LC-MS/MS method for the quantitative estimation of bexicaserin in human plasma and urine. The sample preparation involves the extraction of bexicaserin and internal standard (13CD2-bexicaserin; IS) from 150 μL plasma and 50 μL urine using a solid phase extraction method. The chromatographic separation of bexicaserin and IS was achieved on a Poroshell EC-C18 column using 5 min gradient program. The calibration curve ranged from 0.1 to 100 ng/mL in plasma and 1.0 to 1000 ng/mL in urine. Intraday and interday precision and accuracy, linearity, matrix effect, extraction recovery, carry-over, dilution integrity, a battery of stability studies, and incurred sample reanalysis were performed for bexicaserin in both plasma and urine. The intraday and interday accuracy and precision were well within the acceptable limits in both plasma and urine matrices. Stability studies in plasma and urine showed that bexicaserin was stable on bench for 24 h, in autosampler over 54 h, five freeze-thaw cycles, and long-term storage at -20°C and -80°C for > 368 days. The validated methods were successfully applied in clinical study.
    Keywords:  DEEs; LC–MS/MS; LP352; bexicaserin; human plasma; pharmacokinetics
    DOI:  https://doi.org/10.1002/bmc.6079
  4. Methods Mol Biol. 2025 ;2891 131-152
    Quantitative Lipidomics of Biological Samples Using Supercritical Fluid Chromatography Mass Spectrometry.
    Hiroaki Takeda, Yoshihiro Izumi, Takeshi Bamba.
      Lipidomics has attracted attention in the discovery of unknown biomolecules and for capturing the changes in metabolism caused by genetic and environmental factors in an unbiased manner. However, obtaining reliable lipidomics data, including structural diversity and quantification data, is still challenging. Supercritical fluid chromatography (SFC) is a suitable technique for separating lipid molecules with high throughput and separation efficiency. Here, we describe a quantitative lipidomics method using SFC coupled with mass spectrometry. This technique is suitable for characterizing the structural diversity of lipids (e.g., phospholipids, sphingolipids, glycolipids, and glycerolipids) with high quantitative accuracy to understand their biological functions.
    Keywords:  Cells; Extracellular vesicles; Glycerolipids; Glycolipids; Lipidomics; Lipoproteins; Liquid-liquid extraction; Mass spectrometry; Organs; Phospholipids; Plasma; Quantitative analysis; Sphingolipids; Supercritical fluid chromatography; Tissues
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_7
  5. Anal Chim Acta. 2025 Feb 01. pii: S0003-2670(24)01360-6. [Epub ahead of print]1337 343559
    A sensitive post-column derivatization approach for enhancing hydroxyl metabolites detection.
    Yen-Chu Lin, Shiu-Wen Huang, San-Yuan Wang, Jing-Rong Su, Jimmy Junxiang Wang, Ming-Jen Hsu, Hsiao-Wei Liao.
       BACKGROUND: Chemical derivatization is a common technique in liquid chromatography-mass spectrometry (LC-MS) metabolomics used to improve the ionizability and chromatographic properties of metabolites in complex biological samples. This process facilitates better detection and separation of a wide array of compounds. The reagent 2-(4-boronobenzyl) isoquinolin-2-ium bromide (BBII), developed as a glucose labeling reagent for matrix-assisted laser desorption/ionization MS, enhances ionization for glucose and other hydroxyl metabolites. Its quaternary ammonium group increases ionization efficiency, and its rapid reaction time simplifies pretreatment procedures.
    RESULTS: We developed a novel post-column derivatization (PCD) method using BBII to boost the detection sensitivity of hydroxyl metabolites in LC-MS. By optimizing this BBII PCD approach with 14 hydroxyl-containing compounds, we were able to detect previously undetectable metabolites such as glucose, ribose, and long-chain alcohols. Sensitivity enhancements for these metabolites ranged from 1.1 to 42.9-fold. Applying this method to metabolic profiling of hydroxyl metabolites in the DBTRG-05MG glioblastoma cell line, with and without treatment with the new drug MFB [1-(4-chlorobenzyl)-2-(5-methyl-2-furfurylideneamino)benzimidazole], revealed several hydroxyl metabolites with significantly reduced levels post-treatment.
    SIGNIFICANCE AND NOVELTY: This study presents a new BBII PCD method that substantially improves the detection sensitivity of hydroxyl metabolites in LC-MS. This innovative approach is highly valuable for untargeted metabolomics studies in biological and clinical research, offering a robust tool for identifying metabolite changes and advancing our understanding of metabolic processes in disease and therapeutic contexts.
    Keywords:  2-(4-boronobenzyl) isoquinolin-2-ium bromide; Hydroxyl metabolites; LC-MS; Post-column derivatization; Untargeted metabolomics
    DOI:  https://doi.org/10.1016/j.aca.2024.343559
  6. Methods Mol Biol. 2025 ;2891 109-129
    Untargeted Metabolic Phenotyping by LC-MS.
    Ian D Wilson, Elizabeth Want.
      Untargeted analysis by LC-MS is a valuable tool for metabolic profiling (metabonomics/metabolomics), and applications of this technology have grown rapidly over the past decade. LC-MS offers advantages of speed, sensitivity, relative ease of sample preparation, and large dynamic range compared to other platforms in this role. However, like any analytical approach, there are still drawbacks and challenges that have to be overcome, some of which are being addressed by advances in both column chemistries and instrumentation. In particular, the combination of LC-MS with ion mobility offers many new possibilities for improved analyte separation, detection, and structural identification. There are many untargeted LC-MS approaches which can be applied to metabolic phenotyping, and these usually need to be optimized for the type of sample, the nature of the study, or the biological question. Some of the main LC-MS approaches for untargeted metabolic phenotyping are described in detail in the following protocol.
    Keywords:  Ion mobility; LC-MS; Liquid chromatography; Mass spectrometry; Untargeted metabolic profiling
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_6
  7. Methods Mol Biol. 2025 ;2891 53-66
    Quality Control and Validation Issues in LC-MS-Based Metabolomics.
    Olga Begou, Helen G Gika, Georgios Theodoridis, Ian D Wilson.
      Metabolic profiling performed using untargeted metabolomics of different, complex biological samples aims to apply agnostic/holistic, hypothesis-free, analysis of the small molecules that are present in the analyzed sample. This approach has been the center of major investments and dedicated efforts from the research community for many years. However, limitations and challenges remain, particularly with regard to the validation and the quality of the obtained results. This has led to increasing community engagement, with the formation of think tanks, the establishment of working groups, and the many seminars on quality control (QC) in metabolomics. Here we describe a quality control (QC) protocol used to monitor LC-MS-based metabolomics analysis. A key target is the monitoring of analytical precision. This methodology is described for the analysis of urine but can be applied to different biological matrices, such as various biofluids, cell, and tissue extracts.
    Keywords:  Biological samples; LC-MS; Quality control; Untargeted metabolomics; Validation
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_3
  8. Int J Mol Sci. 2024 Dec 25. pii: 77. [Epub ahead of print]26(1):
    Quantitative Determination of a Series of Oxysterols by an Optimized LC-MS/MS Analysis in Different Tissue Types.
    Zhiting Guo, Huiyan Yu, Kexin Yang, Wenjing Feng, Miao Liu, Tao Wang, Rong Xiao.
      Oxysterols, as metabolites of cholesterol, play a key role in cholesterol homeostasis, autophagosome formation, and regulation of immune responses. Disorders in oxysterol metabolism are closely related to the pathogenesis of neurodegenerative diseases. To systematically investigate the profound molecular regulatory mechanisms of neurodegenerative diseases, it is necessary to quantify oxysterols and their metabolites in central and peripheral biospecimens simultaneously and accurately. However, there are a lot of unsolved problems with the existing methods, such as the hindrance of applying a single method to different biological specimens or the challenge of simultaneous quantification due to differential groups on the ends of the oxysterol side chains. Herein, according to the physicochemical properties and structure of oxysterols, an optimized liquid chromatography-tandem mass spectrometry method for the quantification of oxysterols was established by optimizing the sample preparation process, chromatographic conditions, mobile phase pH, and solvent selection. Seven oxysterols were detected by this method, including 27-hydroxycholesterol, 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol, 7-dehydrocholesterol, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3-hydroxy-5-cholestenoic acid, and 24(S)-hydroxycholesterol. Non-derivatization extraction with methyl tert-butyl ether was used for different biospecimens, followed by simultaneous chromatographic separation of oxysterols on a phenyl hexyl column. By repeated validation, this method exhibited satisfactory linearity, precision, recovery, sensitivity, repeatability, and stability, and it was successfully applied to the detection of oxysterols in the plasma, cerebral cortex, and liver of mouse. In summary, our optimized method enables concurrent analysis and quantification of oxysterols and their metabolites in various biospecimens, presenting a broad range of applicability.
    Keywords:  cerebral cortex; liquid chromatography-tandem mass spectrometry; liver; methyl tert-butyl ether; mouse; non-derivation; oxysterol metabolism; plasma
    DOI:  https://doi.org/10.3390/ijms26010077
  9. Methods Mol Biol. 2025 ;2891 239-256
    Advanced LC-IMS-MS Protocol for Holistic Metabolite Analysis in Wine and Grape Samples.
    Vania Sáez, Sara Ferrero-Del-Teso, Fulvio Mattivi, Urska Vrhovsek, Panagiotis Arapitsas.
      The final aim of metabolomics is the comprehensive and holistic study of the metabolome in biological samples. Therefore, the use of instruments that enable the analysis of metabolites belonging to various chemical classes in a wide range of concentrations is essential, without compromising on robustness, resolution, sensitivity, specificity, and metabolite annotation. These characteristics are crucial for the analysis of very complex samples, such as wine, whose metabolome is the result of the sum of metabolites derived from grapes, yeast(s), bacteria(s), and chemical or physical modification during winemaking. In recent years, a big advantage, in this direction, was the hardware developments on hyphenated instruments that enable the integration of liquid chromatography (LC), ion mobility spectrometry (IMS), and mass spectrometry (MS). This chapter describes an LC-IMS-MS protocol for the analysis of wine and grape samples as well as the use of IMS data in metabolite annotation.
    Keywords:  CCS; HDMSE; Mass spectrometry; Metabolite annotation; Metabolomics; Traveling wave ion mobility; Vitis
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_13
  10. Methods Mol Biol. 2025 ;2891 15-51
    Quality Assurance in Metabolomics and Metabolic Profiling.
    Jennifer A Kirwan, Ulrike Bruning, Jonathan D Mosley.
      Metabolic profiling (untargeted metabolomics) aims for a global unbiased analysis of metabolites in a cell or biological system. It remains a highly useful research tool used across various analytical platforms. Incremental improvements across multiple steps in the analytical process may have large consequences for the end quality of the data. Thus, this chapter concentrates on which aspects of quality assurance can be implemented by a lab in the (pre-)analytical stages of the analysis to improve the overall end quality of their data. The scope of this chapter is limited to liquid-chromatography-mass spectrometry (LC-MS)-based profiling, which is one of the most widely utilized platforms, although the general principles are applicable to all metabolomics experiments.
    Keywords:  Data quality; Mass spectrometry; Metabolomics; NMR; Quality assurance; Quality control; Quality management; Robust method; Untargeted; Validation
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_2
  11. Talanta. 2025 Jan 10. pii: S0039-9140(24)01842-3. [Epub ahead of print]286 127460
    Spatial metabolomics platform combining mass spectrometry imaging and in-depth chemical characterization with capillary electrophoresis.
    Leonidas Mavroudakis, Anastasia Golubova, Ingela Lanekoff.
      Spatial metabolomics offers the combination of molecular identification and localization. As a tool for spatial metabolomics, mass spectrometry imaging (MSI) can provide detailed information on localization. However, molecular annotation with MSI is challenging due to the lack of separation prior to mass spectrometric analysis. Contrarily, surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS) provides detailed molecular information, although the size of the sampling sites is modest. Here, we describe a platform for spatial metabolomics where MSI using pneumatically assisted nanospray desorption electrospray ionization (PA-nano-DESI) is combined with SS-CE-MS to gain both in-depth chemical information and spatial localization from thin tissue sections. We present the workflow, including the user-friendly setup and switching between the techniques, compare the obtained data, and demonstrate a quantitative approach when using the platform for spatial metabolomics of ischemic stroke.
    Keywords:  Capillary electrophoresis; Ischemic stroke; MSI; Spatial metabolomics; Surface sampling
    DOI:  https://doi.org/10.1016/j.talanta.2024.127460
  12. Rapid Commun Mass Spectrom. 2025 Mar 30. 39(6): e9965
    Multiclass Steroid Profiling in Short-Finned Pilot Whale Blubber Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
    Jillian H Wisse, Douglas P Nowacek, Ashley S P Boggs.
       RATIONALE: Wildlife scientists are quantifying steroid hormones in a growing number of tissues and employing novel methods that must undergo validation before application. This study tested the accuracy and precision of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for use on blubber samples from short-finned pilot whales (Globicephala macrorhynchus). We expanded upon a method for corticosteroid quantification by adding analytes and optimizing internal standard (IS) application.
    METHODS: We optimized a method for the quantification of seven steroid hormones using LC-MS/MS with a C18 column. We assessed the accuracy and precision of this updated C18 method and an existing biphenyl method for use with pilot whale blubber by conducting a spike recovery experiment and calculating percent recovery and relative standard deviation (RSD) for each analyte. To explore the potential for running this method with fewer matched ISs, we compared the performance of multiple ISs for each analyte.
    RESULTS: All 11 adrenal and gonadal analytes showed good accuracy and precision in the spike recovery experiment, with recoveries between 82% and 110% and recovery RSDs below 10%. The C18 method detected all analytes at endogenous concentrations, except aldosterone. Although endogenous DHEA was detected, variability was high. IS comparisons showed 10 of 11 analytes could be calculated with comparable accuracy and precision using an IS substitute, but some substitutions significantly altered the analyte concentrations calculated.
    DISCUSSION: The C18 method was not sensitive enough for endogenous aldosterone detection. DHEA, which has not been previously quantified in blubber, was detected in all samples, but with high variability at lower concentrations. The methods in this study provide reliable detection and quantification of the other nine hormones tested and can be used for assessments of adrenal and gonadal steroid hormones from whales. Laboratories can reduce costs through IS substitution but should consider how these substitutions affect results.
    Keywords:  androgens; corticosteroids; liquid chromatography–tandem mass spectrometry; progestogens; steroid hormones
    DOI:  https://doi.org/10.1002/rcm.9965
  13. Methods Mol Biol. 2025 ;2891 257-268
    Analysis of Urinary Metanephrines Using Liquid Chromatography Tandem Mass Spectrometry.
    Marlene N Thaitumu, Elizabeth L Frank.
      Metanephrines (metanephrine [MN] and normetanephrine [NMN]) are O-methylated metabolites derived from the catecholamines, epinephrine, and norepinephrine, respectively. High concentrations of metanephrines have been observed in individuals with pheochromocytoma, a neuroendocrine tumor. Measurement of metanephrines in urine is used to screen for the tumor. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended due to the high sensitivity, specificity, and throughput of the technique. Herein, we describe an optimized LC-MS/MS assay for the analysis of urinary metanephrines.
    Keywords:  Liquid chromatography-tandem mass spectrometry; Metanephrines; Pheochromocytoma
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_14
  14. Anal Chem. 2025 Jan 16.
    OzNOxESI: A Novel Mass Spectrometry Ion Chemistry for Elucidating Lipid Double-Bond Regioisomerism in Complex Mixtures.
    Ryan A Smith, Ashraf M Omar, Fayaj A Mulani, Qibin Zhang.
      Double bond (C═C) position isomerism in unsaturated lipids can indicate abnormal lipid metabolism and pathological conditions. Novel chemical derivatization and mass spectrometry-based techniques are under continuing development to provide more accurate elucidation of lipid structure in finer structural detail. Here, we introduce a new ion chemistry for annotating lipid C═C positions, which is highly efficient for liquid chromatography-mass spectrometry-based lipidomics. This ion chemistry relies on the online derivatization of lipid C═C with ozone and nitrogen oxides upon fragmentation by tandem mass spectrometry, yielding characteristic product ions capable of unambiguously annotating C═C regioisomers. The new workflow was thoroughly evaluated with various glycerophospholipids and fatty acids and applied to human plasma lipid extract, successfully identified and quantified 270 glycerophospholipid and 36 fatty acid C═C isomers with an in-house developed software, OzNOx Companion, for automated data analysis.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05940
  15. Methods Mol Biol. 2025 ;2891 221-237
    UHPLC-TIMS-PASEF®-MS for Lipidomics: From Theory to Practice.
    Vanna Denti, Simone Serrao, Eleonora Bossi, Giuseppe Paglia.
      Trapped ion mobility spectrometry (TIMS) using parallel accumulation serial fragmentation (PASEF®) is an advanced analytical technique that offers several advantages in mass spectrometry (MS)-based lipidomics. TIMS provides an additional dimension of separation to mass spectrometry and accurate collision cross-section (CCS) measurements for ions, aiding in the structural characterization of molecules. This is especially valuable in lipidomics for identifying and distinguishing isomeric or structurally similar compounds. On the other hand, PASEF technology allows for fast and efficient data acquisition by accumulating ions in parallel and then serially fragmenting them. This accelerates the analysis process and improves throughput, making it suitable for high-throughput applications. Moreover, the combination of TIMS and PASEF reduces co-elution and ion coalescence issues, leading to cleaner and more interpretable mass spectra. This results in higher data quality and more confident identifications. In this chapter, a data-dependent TIMS-PASEF® workflow for lipidomics analysis is presented.
    Keywords:  Ion mobility; Lipidomics; Mass spectrometry; PASEF; TIMS
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_12
  16. Anal Chem. 2025 Jan 13.
    Simultaneous Profiling of Multiple Phosphorylated Metabolites in Typical Biological Matrices via Ion-Pair Reversed-Phase Ultrahigh-Performance Liquid Chromatography and Mass Spectrometry.
    Huan Wang, Qinsheng Chen, Yuanpeng Wu, Li Xu, Qi Wang, Qingyu Hu, Huiru Tang.
      Simultaneous analysis of multiple phosphorylated metabolites (phosphorylated metabolome) in biological samples is vital to reveal their physiological and pathophysiological functions, which is extremely challenging due to their low abundance in some biological matrices, high hydrophilicity, and poor chromatographic behavior. Here, we developed a new method with ion-pair reversed-phase ultrahigh-performance liquid chromatography and mass spectrometry using BEH C18 columns modified with hybrid surface technology. This method demonstrated good performances for various phosphorylated metabolites, including phosphorylated sugars and amino acids, nucleotides, NAD-based cofactors, and acyl-CoAs in a single run using standard LC systems. Specifically, the method showed good retention (capacity factor > 2) and reproducibility (ΔtR < 0.09 min, n = 6), peak symmetry (tailing factor < 2), and sensitivity (limit-of-detection < 238 fmol-on-column with QTOFMS) for all tested analytes especially for the medium- and/or long-chain acyl-CoAs. The method demonstrated reproducible applicability across numerous biological matrices, including tissue (liver), human biofluids (urine, plasma), cells, and feces, and revealed significant molecular phenotypic differences in phosphorylated metabolite composition.
    DOI:  https://doi.org/10.1021/acs.analchem.4c04692
  17. Adv Clin Chem. 2025 ;pii: S0065-2423(24)00130-6. [Epub ahead of print]124 123-160
    Ion mobility spectrometry and ion mobility-mass spectrometry in clinical chemistry.
    Kyle E Lira, Jody C May, John A McLean.
      Advancements in clinical chemistry have major implications in terms of public health, prompting many clinicians to seek out chemical information to aid in diagnoses and treatments. While mass spectrometry (MS) and hyphenated-MS techniques such as LC-MS or tandem MS/MS have long been the analytical methods of choice for many clinical applications, these methods routinely demonstrate difficulty in differentiating between isomeric forms in complex matrices. Consequently, ion mobility spectrometry (IM), which differentiates molecules on the basis of size, shape, and charge, has demonstrated unique advantages in the broad application of stand-alone IM and hyphenated IM instruments towards clinical challenges. Here, we highlight representative IM applications and approaches and describe contemporary commercial offerings of IM technology and how these can be, or are currently being, applied to the field of clinical chemistry.
    Keywords:  Breath analysis; High-resolution ion mobility (HRIM); Ion mobility spectrometry (IMS); Ion mobility-mass spectrometry (IM-MS); Lipidomics; Metabolomics; Multidimensional separations; Volatile organic compounds
    DOI:  https://doi.org/10.1016/bs.acc.2024.10.003
  18. Methods Mol Biol. 2025 ;2891 181-204
    HILIC-MS/MS Multi-targeted Method for Metabolomics Applications.
    Christina Virgiliou, Helen G Gika, Georgios Theodoridis.
      Metabolomics aims at identification and quantitation of key end point metabolites, basically polar, in order to study changes in biochemical activities in response to pathophysiological stimuli or genetic modifications. Targeted profiling assays enjoying a growing popularity over the last years with LC-MS/MS as a powerful tool for development of such (semi-)quantitative methods for a large number of metabolites. Here we describe a method for absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, amino acids, nucleotides, organic acids, and amines with a hydrophilic interaction liquid chromatography (HILIC) system comprised with ultra (high) performance liquid chromatography (UHPLC) with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes.
    Keywords:  HILIC-MS/MS; Metabolic profiling; Polar analytes; Targeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_10
  19. Methods Mol Biol. 2025 ;2891 67-89
    Bio- and Chemoinformatic Approaches for Metabolomics Data Analysis.
    Michael Witting, Johannes Rainer.
      Metabolomics data analysis includes, next to the preprocessing, several additional repetitive tasks that can however be heavily dataset dependent or experiment setup specific due to the vast heterogeneity in instrumentation, protocols, or also compounds/samples that are being measured. To address this, various toolboxes and software packages in Python or R have been and are being developed providing researchers and analysts with bioinformatic/chemoinformatic tools to create their own workflows tailored toward their specific needs. This chapter presents tools and example workflows for common tasks focusing on the functionality provided by R packages developed as part of the RforMassSpectrometry initiative. These tasks include, among others, examples to work with chemical formulae, handle and process mass spectrometry data, or calculate similarities between fragment spectra.
    Keywords:  Formula handling; Mass spectra handling; R; RforMassSpectrometry; Spectra similarity calculation
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_4
  20. Anal Chim Acta. 2025 Feb 01. pii: S0003-2670(24)01315-1. [Epub ahead of print]1337 343514
    Enhanced nano-LC-MS for analyzing dansylated oral cancer tissue metabolome dissolved in solvents with high elution strength.
    Ya-Ju Hsieh, Cheng-Yu Hung, Yi-Ting Chen, Yu-Tsun Lin, Kai-Ping Chang, Wei-Fan Chiang, Chih-Yen Chien, Chih-Ching Wu, Liang Li, Jau-Song Yu, Kun-Yi Chien.
       BACKGROUND: Tissue metabolomics analysis, alongside genomics and proteomics, offers crucial insights into the regulatory mechanisms of tumorigenesis. To enhance metabolite detection sensitivity, chemical isotope labeling (CIL) techniques, such as dansylation, have been developed to improve metabolite separation and ionization in mass spectrometry (MS). However, the dissolution of hydrophobic derivatized metabolites in solvents with high acetonitrile content limits the use of liquid chromatography (LC) systems with small-volume reversed-phase (RP) columns. In this study, we established a nano-LC-MS system with an online dilution design to address this issue, enabling sensitive analysis of oral cancer tissue metabolomes.
    RESULTS: Our nano-LC system features a flow path design with online sample dilution before an RP trap column and backflushing of the trap column before entering the analytical column. Compared to other nano-LC systems, both with and without online dilution designs, our system demonstrates the superiority of the T-connector-based dilution method. Using only 1/20th of the sample required for popular micro-LC systems, our nano-LC detects a larger number of peak pairs with similar recovery rates for both hydrophilic and hydrophobic metabolites, ensuring unbiased results. Thirty-two matched pairs of oral squamous cell carcinoma (OSCC) tissue samples and adjacent noncancerous tissues (ANTs) underwent high-throughput CIL-metabolome analysis using our nano-LC system. Compared to our previous micro-LC methods, the nano-LC-MS system exhibits enhanced detection sensitivity, significantly reducing sample requirements.
    SIGNIFICANCE: Our findings highlight the efficacy of our platform for metabolomic analysis with limited sample amounts. The nano-LC system's ability to analyze samples dissolved in strong eluents suggests potential applications for handling other hydrophobic compounds using RPLC or other separation methods facing similar solvent incompatibility issues. This approach holds promise for identifying novel metabolite biomarkers for oral cancers, advancing our understanding of tumorigenesis, and enhancing clinical applications.
    Keywords:  Dansylation; Mass spectrometry; Metabolites; Metabolomics; Nano-LC; Tissue
    DOI:  https://doi.org/10.1016/j.aca.2024.343514
  21. Talanta. 2025 Jan 10. pii: S0039-9140(25)00052-9. [Epub ahead of print]286 127566
    Establishment of a novel large-scale targeted metabolomics method based on NFSWI-DDA mode utilizing HRMS and TQ-MS.
    Rongrong Li, Xinyi Jiao, Xiaolin Wu, Lei Xu, Lin Zhang, Lifeng Han, Guixiang Pai, Wei Mi, Jiang Wu, Liming Wang.
      Metabolites identification is the major bottleneck in untargeted LC-MS metabolomics, primarily due to the limited availability of MS2 information for most detected metabolites in data dependent acquisition (DDA) mode. To solve this problem, we have integrated the iterative, interval, and segmented window acquisition concepts to develop an innovative non-fixed segmented window interval data dependency acquisition (NFSWI-DDA) mode, which achieves comparable MS2 coverage to data independent acquisition (DIA) mode. This acquisition strategy harnesses the strengths of both DDA and DIA, which could provide extensive coverage and excellent reproducibility of MS2 spectra. Furthermore, utilizing the NFSWI-DDA data, we successfully acquired and identified a large-scale of multiple reaction monitoring (MRM) ion pairs, and transitioned them from high-resolution mass spectrometry (HRMS) to triple quadrupole mass spectrometry (TQ-MS). At last, a large-scale targeted metabolomics method was established practically. This method enables targeted analysis of 475 endogenous metabolites encompassing amino acids, nucleotides, bile acids, fatty acids, and carnitines, which could cover 9 major metabolic pathways as well as 65 secondary metabolic pathways. The established targeted method allows for semi-quantitative assessment of 475 metabolites while enabling quantitative analysis of 327 specific metabolites in biological samples. The method demonstrates immense potential in the detection of various biological samples, offering robust technical support and generating extensive data to advance applications in precision medicine and life sciences.
    Keywords:  LC-MS; Large-scale; MRM; NFSWI-DDA; Targeted metabolomics
    DOI:  https://doi.org/10.1016/j.talanta.2025.127566
  22. Methods Mol Biol. 2025 ;2891 269-276
    Two-Phase Extraction for Comprehensive Analysis of the Plant Metabolome by NMR.
    Jan Schripsema, Denise Dagnino.
      Metabolomics is the area of research, which strives to obtain complete metabolic fingerprints, to detect differences between them and to provide hypothesis to explain those differences (Schripsema J, Dagnino D, Handbook of chemical and biological plant analytical methods. Wiley, New York, 2015). However, obtaining complete metabolic fingerprints is not an easy task. Metabolite extraction is a key step during this process, and much research has been devoted to finding the best solvent mixture to extract as much metabolites as possible.Here a procedure is described for analysis of both polar and apolar metabolites using a two-phase extraction system. D2O and CDCl3 are the solvents of choice, and their major advantage is that, for the identification of the compounds, standard databases can be used because D2O and CDCl3 are the solvents most commonly used for pure compound NMR spectra. The procedure enables the absolute quantification of components due to the addition of suitable internal standards. The extracts are also suitable for further analysis with other systems like LC-MS or GC-MS.
    Keywords:  Identification; Metabolic fingerprints; NMR; Plants; Quantification; Two-phase extraction
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_15
  23. Microbiol Resour Announc. 2025 Jan 15. e0099724
    mpactR: an R adaptation of the metabolomics peak analysis computational tool (MPACT) for use in reproducible data analysis pipelines.
    Allison R Mason, Gregory Johnson, Joseph Krampen, Jennifer N T Nguyen, Marcy J Balunas, Patrick D Schloss.
      mpactR automates pre-processing of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data from microbiological samples to correct mispicked peaks, resolve inter-sample variation in abundance across technical replicates, account for in-source ion fragmentation, and remove background noise to yield high-quality mass spectrometry features. The package is available through CRAN and GitHub.
    Keywords:  R package; data curation; metabolomics; software
    DOI:  https://doi.org/10.1128/mra.00997-24
  24. J Sep Sci. 2025 Jan;48(1): e70068
    A Disposable Pipette Extraction-UHPLC-MS/MS Method Based on Removal of Phospholipids to Determine Anandamide, 2-Arachidonoylglycerol, Cannabidiol, and Δ9-Tetrahydrocannabidiol in Plasma Samples.
    Igor Gustavo Carvalho Oliveira, Israel Donizeti de Souza, José Alexandre de Souza de Crippa, Maria Eugênia Costa Queiroz.
      Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the main components of Cannabis sativa plants, can interact with specific cell receptors known as cannabinoid receptors (CBs). The endogenous compounds anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are CB agonists, and, alongside enzymes, they constitute the endocannabinoid system (ECS) and take part in neuromodulation. Several LC-MS/MS methods have been developed to quantify these compounds in biological matrixes, but a fast and simple method that can determine these analytes in plasma samples simultaneously is not available. Here, we propose a disposable pipette extraction technique containing a zirconia-based sorbent (DPX(Zr)) combined with UHPLC-MS/MS analysis to determine CBD, THC, AEA, and 2-AG in plasma samples, simultaneously. The method combines simple protein precipitation (PPT) with a one-step DPX procedure to remove phospholipids, one of the most common endogenous interferents in biological samples. Optimization of the combined PPT-DPX sample preparation method reduced the matrix effect and improved the sensitivity of the analytical method. The validated DPX(Zr)-UHPLC-MS/MS method reported LLOQs of 0.1 ng mL-1 for AEA and 2-AG and 1 ng mL-1 for CBD and THC. The method demonstrated intra- and interassay accuracy and precision of less than 20% for the LLOQ, and less than 15% for the other calibration points. Additionally, no carryover or significant matrix effect was observed. We applied this method to determine AEA, 2-AG, and CBD in plasma samples obtained from obsessive-compulsive disorder patients treated with CBD.
    Keywords:  biological samples; cannabinoids; endocannabinoids; matrix effect; zirconia
    DOI:  https://doi.org/10.1002/jssc.70068
  25. Methods Mol Biol. 2025 ;2891 153-163
    Rat Fecal Metabolomics-Based Analysis.
    Olga Deda, Helen G Gika, Georgios Theodoridis.
      The gut's symbiome, a hidden metabolic organ, has gained scientific interest for its crucial role in human health. Acting as a biochemical factory, the gut microbiome produces numerous small molecules that significantly impact host metabolism. Metabolic profiling facilitates the exploration of its influence on human health and disease through the symbiotic relationship. Fecal metabolomics-based analysis is an indisputably valuable tool for elucidating the biochemistry of digestion and absorption in the gastrointestinal system, serving as the most suitable specimen to study the symbiotic relationship between the host and the intestinal microbiota. It is well-established that the balance of the intestinal microbiota changes in response to various stimuli, both physiological, such as gender, age, diet, and exercise, and pathological, such as gastrointestinal and hepatic diseases. Fecal samples have been analyzed using widely adopted analytical techniques, including NMR spectroscopy, GC-MS, and LC-MS/MS. Rat fecal samples are frequently used and particularly useful substrates for metabolomics-based studies in related fields.The complexity and diversity of fecal samples necessitate careful and skillful handling to extract metabolites, while avoiding their deterioration, effectively and quantitatively. Several determinative factors, such as the fecal sample weight to extraction solvent solution volume, the nature and pH value of the extraction solvent, and the homogenization process, play crucial roles in achieving optimal extraction for obtaining high-quality metabolic fingerprints, whether for untargeted or targeted metabolomics.
    Keywords:  Fecal extract; Fecal samples; GC-MS; Gut microbiome; Gut microbiota; LC-MS/MS; Metabolic profiling; Metabolites; Metabolome; Metabolomics; Metabonomics; NMR; Rats; Sample preparation
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_8
  26. J Pharm Biomed Anal. 2025 Jan 09. pii: S0731-7085(25)00011-1. [Epub ahead of print]256 116670
    Hydrophilic interaction liquid chromatography-tandem mass spectrometry analysis of oligosaccharides and iridoid glycosides in rat plasma: Pharmacokinetic characterization of previously overlooked oligosaccharides from Radix Rehmanniae.
    Hanyang Wang, Yue Du, Chunwei Zhou, Yan Wang, Xin Wang.
      Radix Rehmanniae (RR) is a widely used herb in traditional Chinese Medicine with properties of tonifying the kidneys and nourishing the blood. Both raw and processed RR are effective for the treatment of diabetes in clinical practice. Oligosaccharides and iridoid glycosides are the primary active components responsible for the anti-diabetic effects of RR. In this study, a rapid and sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for simultaneous determination of oligosaccharides (raffinose, manninotriose and stachyose) and iridoid glycosides (catalpol and ajugol) in rat plasma. Significant analytical challenges were encountered during method development, including distinct retention behaviors of oligosaccharides and iridoid glycosides, low ionization and extraction efficiency of oligosaccharides, thermal instability of catalpol and reduced column performance. The strategies to overcome these challenges were presented by optimizing chromatographic separation, mass spectrometric detection and sample preparation. The best separation was achieved using an Accucore-150-Amide-HILIC column (100 mm × 2.1 mm, 2.6 μm) at 50 °C with mobile phase consisted of acetonitrile and ammonium acetate (2.5 mM) under gradient elution. Ammonium adduct ions produced by positive electrospray ionization were chosen as precursor ions for multiple reaction monitoring transitions. The established HILIC-MS/MS method exhibited good linearity (r > 0.9937) with the lower limits of quantification of 0.01-0.2 μg/mL using only 50 µL of plasma sample. The method was successfully applied to pharmacokinetic characterization of oligosaccharides and iridoid glycosides in normal and type 2 diabetic rats following intragastric administration of raw and processed RR extracts.
    Keywords:  Hydrophilic interaction chromatography; Iridoid glycosides; Oligosaccarides; Pharmacokinetics; Radix Rehmanniae; Tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.jpba.2025.116670
  27. J Pharm Biomed Anal. 2025 Jan 08. pii: S0731-7085(25)00013-5. [Epub ahead of print]256 116672
    A reliable LC-MS/MS method for the quantification of natural amino acids in human plasma and its application in clinic.
    Junhuan Lin, Yibo Song, Yangrui Zhang, Tao Ke, Fengting Ou, Kui Zeng, Debo He, Li Li, Lushan Yu.
      A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 L-amino acids (AAs) in human plasma. Chromatographic separation was achieved on an Agilent AdvanceBio Hilic column within 15 min via gradient elution with an aqueous solution containing 5 mM ammonium formate, 5 mM ammonium acetate and 0.1 % formic acid and an organic mobile phase containing 0.1 % formic acid, 5 mM ammonium formate and 5 mM ammonium acetate acetonitrile-water (90:10, v/v) at the flow rate of 0.25 mL/min. Individual AAs and internal standard were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this LC-MS/MS method as a specific, accurate, and reliable assay. The method was thus utilized to compare the dynamics of individual plasma AAs between healthy females and patients with ovarian tumors. Our results revealed that, in cancer group, plasma 3-Methyl-L-Histidine, L-Proline, L-Phenylalanine and L-Lysine concentrations were significantly increased in patients with malignant ovarian tumors while L-Leucine and L-Isoleucine levels were sharply decreased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for ovarian cancer.
    Keywords:  Amino acids; Biomarker; Ovarian cancer progression; UHPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.jpba.2025.116672
  28. bioRxiv. 2024 Oct 02. pii: 2024.09.25.614780. [Epub ahead of print]
    A Differential Ion Mobility Acoustic Ejection Mass Spectrometer System for Screening Isomerization-Mediating Enzyme Drug Targets.
    Samad Bazargan, Patricia Dranchak, Chang Liu, James Inglese, John Janiszewski, Bradley B Schneider, Thomas R Covey.
      We report the first implementation of ion mobility mass spectrometry combined with an ultra-high throughput sample introduction technology for high throughput screening (HTS). The system integrates differential ion mobility (DMS) with acoustic ejection mass spectrometry (AEMS), termed DAEMS, enabling the simultaneous quantitation of structural isomers that are the sub-strates and products of isomerase mediated reactions in intermediary metabolism. We demonstrate this potential by comparing DAEMS to a luminescence assay for the isoform of phosphoglycerate mutase (iPGM) distinctively present in pathogens offering an opportunity as a drug target for a variety of microbial and parasite borne diseases. The metabolome consists of many structural isomers that require for separation a mobility resolving power of more than 300. Resolving powers measured in collision cross section space of 1588 and 1948 for 2- and 3-phosphoglycerate and the citrate/isocitrate isomeric pairs respectively are shown. These are the highest reported ion mobility resolving powers for molecules from the metabolome reported to date. The potential for DAEMS as a generalized screening tool is demonstrated with the separation of the substrates and products of two additional isomerases that present as potential therapeutic targets, chorismate mutase and triosephosphate isomerase. The separations are achieved at speeds compatible with the sample introduction rates of AEMS providing sufficient data points to integrate the peaks for quantitation without the use of internal standards. DMS hyphenated with acoustic sample ejection MS provides a unique solution to high throughput mass spectrom-etry applications where isomer and other types of separations are required.
    DOI:  https://doi.org/10.1101/2024.09.25.614780
  29. Am J Transl Res. 2024 ;16(12): 8008-8022
    Determination of osimertinib concentration in rat plasma and lung/brain tissues.
    Yalan Zuo, Peidan Yang, Ruixia Yang, Juan Hou, Rui Feng, Ping Liang, Jiang Liu.
       OBJECTIVES: The aim of this study was to establish an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the detection of osimertinib in rat plasma, lung and brain tissues.
    METHODS: Forty-eight rats were randomly divided into an experimental group (receiving osimertinib at doses of 5, 8, and 10 mg/kg) and a control group. After continuous intragastric administration for 15 days, samples of blood, lung, and brain tissue were collected. Chromatographic separation was achieved using a BEH C18 column with gradient elution, employing a mobile phase of water (containing 0.1% (v/v) formic acid) and acetonitrile. The concentration of osimertinib in the samples was determined using an AB SCIEX 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) mode.
    RESULTS: A UPLC-MS/MS analytical method for determining osimertinib concentrations was successfully established and validated. A linear relationship was observed for osimertinib concentrations in plasma within the range of 1-300 ng/mL, and in lung and brain tissues within the range of 0.5-50 ng/mL. The selectivity, accuracy, precision, matrix effect, extraction recovery, and stability all meet the requirements of methodological validation criteria.
    CONCLUSIONS: A rapid and sensitive UPLC-MS/MS method was developed and validated for quantifying osimertinib concentrations in rat plasma, lung, and brain tissues, providing a valuable tool for pharmacokinetic and tissue distribution studies.
    Keywords:  EGFR-tyrosine kinase inhibitor; Osimertinib; UPLC-MS/MS; concentration determination
    DOI:  https://doi.org/10.62347/SYZD2489
  30. J Chromatogr A. 2025 Jan 03. pii: S0021-9673(24)01023-9. [Epub ahead of print]1742 465650
    Enhancing lipid identification in LC-HRMS data through machine learning-based retention time prediction.
    Hamada A A Noreldeen.
      The comprehensive identification of peaks in untargeted lipidomics using LC-MS/MS remains a significant challenge. Confidence in lipid annotation can be greatly improved by integrating a highly accurate machine learning-based retention time prediction model. Such an approach enables the identification of lipids for understanding pathogenic mechanisms, biomarker discovery, and drug screening. In this study, we developed a machine learning model to predict retention times and facilitate lipid peak annotations in LC-MS-based untargeted lipidomics. Our model achieved high correlation coefficients of 0.998 and 0.990, with mean absolute errors (MAE) of 0.107 min and 0.240 min for the training and test sets, respectively. External validation showed similarly strong performance, with correlations of 0.991 and 0.978, and MAE values of 0.241 min and 0.270 min. We also compared the impact of molecular descriptors and molecular fingerprints on the model's performance, finding that molecular descriptors outperformed molecular fingerprints across all datasets when using Random Forest (RF) for model construction. Notably, this retention time calibration model demonstrates robust performance across chromatographic systems with comparable gradients and flow rates. Overall, this machine learning model enhances lipid annotation accuracy and reduces errors in untargeted lipidomics, improving data analysis across multiple datasets.
    Keywords:  LC-high resolution MS; Lipid identification; Machine learning; Random forest; Retention time prediction model
    DOI:  https://doi.org/10.1016/j.chroma.2024.465650
  31. Anal Chim Acta. 2025 Feb 01. pii: S0003-2670(24)01353-9. [Epub ahead of print]1337 343552
    A novel 3D-printed sample preparation method for benzodiazepine quantification in human serum.
    Dagmara Kroll, Szymon Ulenberg, Paweł Georgiev, Bartosz Marciniak, Gert Desmet, Tomasz Bączek, Mariusz Belka.
       BACKGROUND: Benzodiazepine abuse remains a significant public health concern. Current sample preparation methods for benzodiazepine analysis from human serum often involve complex procedures that require large sample volumes and extensive organic solvent use. To address these limitations, this study presents a novel and efficient sample preparation method utilizing 3D-printed sorbent devices.
    RESULTS: The 3D-printed devices, fabricated from a thermoplastic composite incorporating C18-modified silica, demonstrated exceptional performance in extracting benzodiazepines from human serum. The method was optimized and validated according to ICH guidelines, ensuring its reliability for quantitative benzodiazepine analysis. Notably, the method required minimal sample and solvent volumes, eliminating the need for protein precipitation, evaporation, and reconstitution.
    SIGNIFICANCE: This novel sample preparation approach offers significant advantages over traditional methods, providing a more efficient and environmentally friendly solution for benzodiazepine analysis. The versatility of 3D printing allows for the customization of sorbent devices for various analytes and matrices, expanding the potential applications of this method. Coupled with a rapid and robust LC-MS method optimized with DryLab, this approach presents a valuable and sensitive tool for benzodiazepine monitoring in clinical and toxicological settings.
    Keywords:  3D printing; Benzodiazepines; Fused deposition modelling; Human serum; Sample preparation; Solid-phase microextraction
    DOI:  https://doi.org/10.1016/j.aca.2024.343552
  32. J Chem Inf Model. 2025 Jan 16.
    ABCoRT: Retention Time Prediction for Metabolite Identification via Atom-Bond Co-Learning.
    Guangbin Cheng, Bingyi Wang, Nannan Bai, Weihua Li.
      Liquid chromatography retention time (RT) prediction plays a crucial role in metabolite identification, a challenging and essential task in untargeted metabolomics. Accurate molecular representation is vital for reliable RT prediction. To address this, we propose a novel molecular representation learning framework, ABCoRT(Atom-Bond Co-learning for Retention Time prediction), designed for predicting metabolite retention times. Our model transforms molecular graphs into dual hypergraphs, enabling the collaborative updating of atomic and bond information within both molecular graphs and hypergraphs, thereby producing highly informative molecular representations. We evaluated ABCoRT on a large-scale Small Molecule Retention Time (SMRT) data set comprising 80,038 molecules. Our model achieved a mean absolute error (MAE) of 25.75 s and a mean relative error (MRE) of 3.24% after removing nonretained molecules. Additionally, we fine-tuned pretrained ABCoRT models on six additional data sets from PredRet, achieving the lowest MAEs on five of them. Additionally, in metabolite screening conducted on the MetaboBASE and RIKEN_PlaSM data sets from the MassBank of North America, ABCoRT demonstrates its capability to filter out 38.35 and 28.46% of candidate compounds, respectively.
    DOI:  https://doi.org/10.1021/acs.jcim.4c02179
  33. J Biomed Mater Res A. 2025 Jan;113(1): e37864
    A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.
    Samuel J Rozans, Yingjie Wu, Abolfazl S Moghaddam, E Thomas Pashuck.
      Peptides are widely used in biomaterials due to their ease of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell-secreted enzymes, which creates the possibility of utilizing cell-secreted enzymes for tuning cell-material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography-mass spectrometry (LC-MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC-MS for the multiplexed quantification of cell-induced peptide degradation, including the need for nondegradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. Removing these constituents can be expensive, time-consuming, and increases sample variability. However, loading unpurified samples onto the column without removing lipids and proteins will foul the column. Here, we show that directly injecting complex, unpurified samples onto the LC-MS without any purification enables rapid and accurate quantification of peptide concentration and that hundreds of LC-MS runs can be done on a single column without significantly diminishing the ability to quantify the degradation of peptide libraries. To understand how repeated injections degrade column performance, a model library was injected into the LC-MS hundreds of times. It was then determined that column failure is evident when hydrophilic peptides are no longer retained on the column and that failure can be easily identified by using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost-effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide-based therapies.
    DOI:  https://doi.org/10.1002/jbm.a.37864
  34. J Sep Sci. 2025 Jan;48(1): e70079
    A Novel Approach to the Isolation of Disease Markers: Quantitative Analysis of Amino Acids in Exhaled Breath Condensates from Sleep Apnea Patients with Enhanced Sampling and LC-ESI-MS.
    Anna Kaliszewska, Piotr Struczyński, Sylwia Kwiatkowska, Daruisz Nowak, Tomasz Bączek, Lucyna Konieczna.
      Interest in obstructive sleep apnea is rising due to its neurocognitive and cardiovascular impacts, including systemic hypertension, myocardial infarction, and cerebrovascular events. Obstructive sleep apnea diagnosis can be suggested through symptoms like snoring, daytime sleepiness, and physical signs like increased neck circumference; however, overnight polysomnography is recommended to confirm. Exhaled breath condensate has emerged as a novel, noninvasive technique for biomarker sample collection. It is simple, rapid, repeatable, and suitable for young children and severely ill patients. A direct method using liquid chromatography-mass spectrometry coupled with hydrophilic interaction chromatography was established for quantitative analysis of amino acids in human exhaled breath condensate samples. The separation was performed using XBridge Amide BEH column and a mobile phase of ammonium formate buffer in gradient conditions. The method exhibited a low detection limit (0.08-1.28 ng/mL), good linearity (R2 between 0.9909 and 0.9987), and high recoveries (93-101.3%) for 21 studied amino acids with interday RSD of 2.1-7.7%. The LC-MS method was verified and applied to determine amino acids in exhaled breath condensate samples from obstructive sleep apnea patients, offering fast, reliable analysis without derivatization as a noninvasive alternative to standard methods.
    Keywords:  LC‐MS; amino acids; exhaled breath condensates; sleep apnea
    DOI:  https://doi.org/10.1002/jssc.70079
  35. Molecules. 2024 Dec 31. pii: 121. [Epub ahead of print]30(1):
    A Novel Liquid Chromatography-Tandem Mass Spectrometry Method to Quantify Tryptophan and Its Major Metabolites in Serum to Support Biomarker Studies in Patients with Cancer Undergoing Immunotherapy.
    Anna Siemiątkowska, Barbara Kuźnar-Kamińska, Katarzyna Kosicka-Noworzyń, Kamila Nowaczewska, Hanna Winiarska, Dominika Popiołek, Filip Kamiński, Franciszek K Główka.
      Tryptophan (TRP) is an essential amino acid crucial for the production of many bioactive compounds. Disturbances in TRP metabolism have been revealed in various diseases, many of which are closely related to the immune system. In recent years, we have focused on finding blood-based biomarkers of successful immunotherapy in cancer. Thus, we aimed to develop a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for TRP and its metabolites that could be used in biomarker studies. Although analyzing TRP derivatives in biological matrices is not a new topic, we encountered multiple challenges during method development. One of them was the phenomenon of cross-interferences between the analyzed molecules, which has not been explored in most published papers. We noticed that injecting a pure single-compound solution often generated a signal in the other compounds' MS/MS channels. Specifically, TRP generated unexpected peaks in the channel for kynurenine, kynurenic acid, and xanthurenic acid, while kynurenine generated peaks in the channel for kynurenic acid. We also recorded a mutual cross-talk between kynurenine and isotope-labeled TRP. Different origins of the observed cross-signal contribution were proposed. This paper draws attention to investigating cross-interferences in LC-MS/MS, especially when structurally related compounds will be analyzed. Despite all the challenges, the method was successfully validated according to international guidelines (EMA/ICH), and its applicability was confirmed in a pilot study including 20 patients with lung cancer undergoing chemoimmunotherapy.
    Keywords:  cross-interferences; cross-signal contribution; cross-talk; poor analyte stability; similar MRMs; standard impurity
    DOI:  https://doi.org/10.3390/molecules30010121
  36. Anal Chem. 2025 Jan 16.
    Development and Validation of a Highly Sensitive Isotope-Coded Equivalent Reporter Ion Assay for the Semi-Quantification of Isocoumarins in Complex Matrices.
    Xiao-Feng Huang, Peng-Yu Liang, Xiao-Dan Yin, Yue-Cai Hou, Tian-Tian Wang, Ruo-Wen Li, Zhifeng Zhang, Xiaojun Yao, Pei Luo, Lin-Sen Qing.
      The accurate quantification of multicomponents using LC-MS is pivotal for ensuring the quality control of herbal medicine, as well as the investigation of their analysis of biological tissue distribution. However, two significant challenges persist: the scarcity of authentic standards and the selection of appropriate internal standards. In this study, we present a highly sensitive isotope-coded equivalent reporter ion assay (iERIA) that combines equivalently quantitative ion and isotope-coded derivatization strategies. This method offers triple functionality: enabling the semidetermination of multiple components using a single standard, introducing stable isotope-labeled internal standards, and enhancing MS detection signals. Using four isocoumarins as a model, namely, 5-carboxylmellein, 5-hydroxymethylmellein, 5-methylmellein, and 5-hydroxymellein, we successfully quantified these compounds across various matrices, including herbal extracts, plasma, urine, and liver tissue. Reporter ions at m/z 170 and 234 generated by the dansulfonyl derivatives of isocoumarins, were subsequently detected for calculating the concentrations of samples based on the equivalent ion method. It is very beneficial for trace detection in biological samples free of any concentration steps, with an increased LOD of 50 times after dansyl chloride derivatization. Additionally, the introduction of stable isotope-labeled internal standards using d6-dansyl chloride mitigated matrix effects and instrument drift, ensuring the accuracy and precision of the semiquantification. This practical UPLC-MS/MS strategy significantly expands the applicability of multicomponent determination, with promising implications in diverse domains such as herbal medicine active ingredient analysis, food function and safety assessment, and metabolomics research.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05069
  37. J Chromatogr A. 2025 Jan 25. pii: S0021-9673(24)00983-X. [Epub ahead of print]1741 465610
    Separation of polar and neutral lipids from mammalian cell lines by high-performance thin-layer chromatography.
    Ana Rita Dias Araújo, Océane Buvry, Bruno Antonny, Delphine Debayle.
      The introduction of high-performance TLC (HPTLC) instrumentation that allows precise control of critical parameters has transformed the technique into an efficient and rapid tool for analyzing various metabolites, namely lipids. Although mass spectrometry (MS) has largely replaced lipid analysis techniques over recent decades due to its comprehensive lipidome profiling capabilities, it typically lacks the rapidity and simplicity of TLC. HPTLC remains advantageous due to its ease of use, simpler data interpretation, and compatibility with complementary techniques. In this study, we established a HPTLC protocol to fractionate both polar and non-polar lipids on a single normal phase plate. Twenty lipid standards were fractionated and the method was successfully applied to whole extracts from six mammalian cell lines. Standards and extracted lipids were applied with an automated sampler, and polar lipids were first fractionated in a 5-step automated gradient elution, followed by the fractionation of neutral lipids in a twin-trough chamber with three different elutions. Plates were automatically sprayed with a modified copper sulfate solution and charred to reveal lipids and obtain the respective chromatograms. LC-MS was used to identify ambiguous bands, thus ensuring the accuracy of lipid identification.
    Keywords:  HPTLC; Lipid fractionation
    DOI:  https://doi.org/10.1016/j.chroma.2024.465610
  38. BMC Bioinformatics. 2025 Jan 10. 26(1): 8
    BiomiX, a user-friendly bioinformatic tool for democratized analysis and integration of multiomics data.
    PRECISESADS Flow Cytometry Study Group, PRECISESADS Clinical Consortium
       BACKGROUND: Interpreting biological system changes requires interpreting vast amounts of multi-omics data. While user-friendly tools exist for single-omics analysis, integrating multiple omics still requires bioinformatics expertise, limiting accessibility for the broader scientific community.
    RESULTS: BiomiX tackles the bottleneck in high-throughput omics data analysis, enabling efficient and integrated analysis of multiomics data obtained from two cohorts. BiomiX incorporates diverse omics data, using DESeq2/Limma packages for transcriptomics, and quantifying metabolomics peak differences, evaluated via the Wilcoxon test with the False Discovery Rate correction. The metabolomics annotation for Liquid Chromatography-Mass Spectrometry untargeted metabolomics is additionally supported using the mass-to-charge ratio in the CEU Mass Mediator database and fragmentation spectra in the TidyMass package. Methylomics analysis is performed using the ChAMP R package. Finally, Multi-Omics Factor Analysis (MOFA) integration identifies shared sources of variation across omics data. BiomiX also generates statistics, report figures and integrates EnrichR and GSEA for biological process exploration and subgroup analysis based on user-defined gene panels enhancing condition subtyping. BiomiX fine-tunes MOFA models, to optimize factors number selection, distinguishing between cohorts and providing tools to interpret discriminative MOFA factors. The interpretation relies on innovative bibliography research on Pubmed, which provides the articles most related to the discriminant factor contributors. Furthermore, discriminant MOFA factors are correlated with clinical data, and the top contributing pathways are explored, all with the aim of guiding the user in factor interpretation.
    CONCLUSIONS: The analysis of single-omics and multi-omics integration in a standalone tool, along with MOFA implementation and its interpretability via literature, represents significant progress in the multi-omics field in line with the "Findable, Accessible, Interoperable, and Reusable" data principles. BiomiX offers a wide range of parameters and interactive data visualization, allowing for personalized analysis tailored to user needs. This R-based, user-friendly tool is compatible with multiple operating systems and aims to make multi-omics analysis accessible to non-experts in bioinformatics.
    Keywords:  Data integration; MOFA; Multiomics; User-friendly
    DOI:  https://doi.org/10.1186/s12859-024-06022-y
  39. J Chromatogr Sci. 2025 Jan 14. pii: bmaf001. [Epub ahead of print]63(2):
    Quality by Design-Steered Chromatographic Separation and Identification of the Geometric Isomers of Capsiate by Reversed-Phase HPLC and LC-MS.
    Reena Gupta, Bhupinder Kapoor, Monica Gulati, Sumant Saini, Pooja Rani, Atanas G Atanasov, Stefano Dall'Acqua, Qushmua Alzahrani, Deepak Kapoor, Jesus Simal-Gandara.
      An efficient reverse-phase high-performance liquid chromatographic method, based on the design of the experiment approach, was developed for the simultaneous determination of capsiate isomers. Critical method parameters, i.e., flow rate and mobile phase composition, demarcated during preliminary screening were optimized using central composite design. Chromatographic separation was achieved on a Nucleodur C18 column (250 × 4.6 mm, 5 μm), with the mobile phase consisting of water-acetonitrile (40:60), both acidified with 0.1% v/v formic acid, passed at a flow rate of 1 mL/min. E- and Z-capsiates were eluted from the column at the retention times of 18.56 ± 0.09 and 17.30 ± 0.08 min, respectively, with a resolution factor of 1.693 ± 0.046. The method was found to be linear within the concentration range of 1.054-5.270 and 8.623-43.115 μg/mL for Z- and E-isomers, respectively, with an R2 of >0.99. Recovery of the individual values was in range of 96.15-101.92%, with a relative standard deviation of <2%. The developed method was used to quantify capsiate isomers extracted from sweet pepper fruits. Therefore, the proposed method is presented for optimum isomeric resolution of these closely related E- and Z-isomers with convenient sample preparation, acceptable run-time, cost effectiveness and use of conventional instruments.
    HIGHLIGHTS:
    DOI:  https://doi.org/10.1093/chromsci/bmaf001
  40. Mass Spectrom Rev. 2025 Jan 10.
    Progress in Tandem Mass Spectrometry Data Analysis for Nucleic Acids.
    Michael B Lanzillotti, Jennifer S Brodbelt.
      Mass spectrometry (MS) has become a critical tool in the characterization of covalently modified nucleic acids. Well-developed bottom-up approaches, where nucleic acids are digested with an endonuclease and the resulting oligonucleotides are separated before MS and MS/MS analysis, provide substantial insight into modified nucleotides in biological and synthetic nucleic. Top-down MS presents an alternative approach where the entire nucleic acid molecule is introduced to the mass spectrometer intact and then fragmented by MS/MS. Current top-down MS workflows have incorporated automated, on-line HPLC workflows to enable rapid desalting of nucleic acid samples for facile mass analysis without complication from adduction. Furthermore, optimization of MS/MS parameters utilizing collision, electron, or photon-based activation methods have enabled effective bond cleavage throughout the phosphodiester backbone while limiting secondary fragmentation, allowing characterization of progressively larger (~100 nt) nucleic acids and localization of covalent modifications. Development of software applications to perform automated identification of fragment ions has accelerated the broader adoption of mass spectrometry for analysis of nucleic acids. This review focuses on progress in tandem mass spectrometry for characterization of nucleic acids with particular emphasis on the software tools that have proven critical for advancing the field.
    DOI:  https://doi.org/10.1002/mas.21923
  41. Arch Toxicol. 2025 Jan 17.
    Exploring the potential of MRM-IDA-EPI method in mass spectrometry for exposomic analysis: a commentary.
    Ravikumar Jagani, Jasmin Chovatiya, Hiraj Patel, Syam S Andra.
      Exposomics is a field that studies environmental exposures and their impact on human health. The MRM-IDA-EPI method, which combines targeted and untargeted mass spectrometry methods, is useful for identifying and quantifying biomarkers in various biological matrices. The method's accuracy and precision in forensic toxicological screening suggest potential applications for detecting low-level environmental exposures. It can help detect and understand environmental exposures, explain their metabolic processes, and assess their impact on human health more effectively.
    Keywords:  Biomonitoring; Exposome; Hybrid Targeted/Untargeted Screening; MRM-IDA-EPI Scan; QTRAP LC-MS/MS
    DOI:  https://doi.org/10.1007/s00204-025-03958-9
  42. Anal Chem. 2025 Jan 12.
    Microseparation of Lipophilic and Hydrophilic Metabolites for Single Oocyte Mass Spectrometry Analysis.
    Jinlei Yang, Jing Wang, Runsong Cheng, Tianyi Liao, Siyuan Pan, Murong Du, Weiliang Liu, Liying Yan, Sichun Zhang, Xinrong Zhang.
      Single-cell metabolic analysis has not yet achieved the coverage of bulk analysis due to the diversity of cellular metabolites and the ionization competition among species. Direct ionization methods without separation lead to the masking of low-intensity species. By designing a capillary column emitter and introducing reverse-phase chromatography principles, we achieved the microseparation of lipophilic and hydrophilic metabolites and lowered the limit of detection of hydrophilic metabolites to the level of a single oocyte. We identified 517 metabolite species in a single oocyte, achieving coverage and reproducibility comparable to those of bulk analysis. By comparing oocytes at different maturation stages, 76 metabolic features were identified with significant differences between the germinal vesicle and meiosis II stages. Metabolite level changes suggested the roles of lipid metabolism remodeling, increased amino acid synthesis, and a shift from pyrimidine metabolism to purine metabolism in the process of oocyte maturation. This microseparation mass spectrometry analysis is expected to promote single-cell metabolomics.
    DOI:  https://doi.org/10.1021/acs.analchem.4c04900
  43. Anal Chim Acta. 2025 Feb 01. pii: S0003-2670(24)01363-1. [Epub ahead of print]1337 343562
    Composition-dependent MRM transitions and structure-indicative elution segments (CMTSES)-based LC-MS strategy for disaccharide profiling and isomer differentiation.
    Wen Miao, Na Li, Jia-Qian Chen, Jian-Lin Wu.
       BACKGROUND: Carbohydrates exhibit diverse functions and extensive biological activities and are notable in the field of life sciences. However, their inherent diversity and complexity-steaming from variations in isomeric monomers, glycosidic bonds, configurations, etc.-present considerable challenges in structural analysis. Considering these challenges, the disaccharide building blocks with simpler structures could provide more structural information. Although various approaches have been explored, sufficient standards or specialized equipment are required to differentiate and characterize isomers. Therefore, a strategy that addresses these challenges is urgently needed.
    RESULTS: A Composition-dependent MRM Transitions and Structure-indicative Elution Segments (CMTSES)-based liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS) strategy was developed to comprehensively profile disaccharide units and differentiate isomers. First, the composition-related precursor and structure-specific product ions of disaccharides were generated by QQQ-MS. Thereout, MRM transitions were proposed to enable the comprehensive profiling of disaccharides and rapid annotation of their compositions and saccharide types at both termini. Next, the linkage, composition, and configuration isomers of disaccharides were effectively differentiated and presented characteristic LC elution. Furthermore, low-cost and available "location references" (mannose, galactose, and isomaltose) were sought to define structure-indicative elution segments for the identification of isomeric hexose disaccharides. Building on this foundation, the novel CMTSES-based LC-MS strategy was designed, and its feasibility was further verified by successfully differentiating and identifying mixed homogenous and/or heterogenous disaccharide isomers in real samples. Sufficient structural information was obtained even for those consisting of diversified monomer types.
    SIGNIFICANCE AND NOVELTY: This strategy comprehensively profiles both major and minor disaccharides and effectively differentiates multiple types of isomers. The use of readily available "location references" facilitated the identification of isomeric hexose disaccharide with reduced dependence on standards, thereby broadening the applicability of this strategy. However, the characterization of disaccharides with other compositions is challenging. Further in-depth investigations into intramolecular hydrogen bond simulation should provide solutions. Additionally, CMTSES-based LC-MS strategy is promising to analyze complex structures and samples.
    Keywords:  CMTSES-based LC-MS strategy; Characteristic ions-based MRM transition prediction; Complex structure or sample analysis; Disaccharide profiling; Isomer differentiation; Structure-indicative LC elution segments
    DOI:  https://doi.org/10.1016/j.aca.2024.343562
  44. Anal Chem. 2025 Jan 13.
    iTASO: A Novel Photosensitive Probe for High-Throughput and Selective Submetabolomic Analysis via Flow Injection-Mass Spectrometry.
    Pei-Rong Bai, Na An, Yan-Zhen Wang, Yao-Yu Chen, Quan-Fei Zhu, Yu-Qi Feng.
      Flow injection mass spectrometry (FI-MS) is widely employed for high-throughput metabolome analysis, yet the absence of prior separation leads to significant matrix effects, thereby limiting the metabolome coverage. In this study, we introduce a novel photosensitive MS probe, iTASO-ONH2, integrated with FI-MS to establish a high-throughput strategy for submetabolome analyses. The iTASO probe features a conjugated-imino sulfonate moiety for efficient photolysis under 365 nm irradiation and a reactive group for selective metabolite labeling. The iTASO-ONH2 probe effectively and selectively labels carbonyl compounds, forming highly stable labeled products. Upon UV exposure, the labeled products rapidly release sulfonic acid-containing photolysis products, detectable with high sensitivity in ESI-negative mode and low matrix effect, offering femtomole-level detection sensitivity. The iTASO-ONH2-based FI-MS strategy was applied to fecal samples from chronic sleep-deprived and control mice, revealing 192 potential carbonyl compounds of which 37 exhibited significant alterations. Additionally, three other photosensitive probes─iTASO-NH2, iTASO-NHS, and iTASO-MAL─were synthesized to selectively label carboxyl, amino, and thiol metabolites, respectively, underscoring the versatility of the iTASO-based FI-MS strategy for submetabolomic analysis across diverse metabolite classes.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06108
  45. J Anal Toxicol. 2025 Jan 13. pii: bkae100. [Epub ahead of print]
    Beyond the baseline: Quantification of two Phosphatidylethanol (PEth) homologues in Whole Blood by LC-MS/MS and Retrospective Data Analysis from a National Reference Laboratory.
    Nicole J Mathewson, Nkemakonam C Okoye, Heather A Nelson, Vrajesh Pandya, Chad Moore, Kamisha L Johnson-Davis.
      Background - Alcohol is the most abused substance in Western society, resulting in major economic losses and negative health consequences. Therefore, there is a need for a selective and robust detection method for alcohol consumption in various clinical and forensic settings. This study aimed to validate a mass spectrometry method for quantifying phosphatidylethanol (PEth) and perform retrospective data analysis from the patient population of a national reference laboratory.
    METHODS: Quantification of phosphatidylethanol in whole blood was accomplished using an LC-MS/MS assay. Isotopically labeled internal standard for the two PEth homologues was added to the whole blood specimen, followed by protein precipitation with a mixture of acetonitrile and isopropyl alcohol. After centrifugation, an aliquot of the supernatant was buffered with ammonium acetate before LC-MS/MS analysis on an Agilent 6470 triple quadrupole mass spectrometer coupled to an Agilent 1260 Infinity II LC system. This LC-MS/MS assay was validated for clinical use in accordance with CLSI guidelines.
    RESULTS: The analytical measurement range, 10 - 2000 ng/mL, was linear with R2 of 0.999. The within-run and total imprecision was < 5% CV for the low (20 ng/mL), medium (200 ng/mL), and high QC (1000 ng/mL). Results from accuracy and method comparison experiments met the bias criteria of ±15%. Retrospective data analysis showed ~27% of patients had PEth concentrations greater than 20 ng/mL. Males and females had similar positivity rates for PEth and the positivity rate of women of reproductive age (15-44 years old) was 35% in comparison to 25% in women 45-89 years old.
    CONCLUSIONS: This study's LC-MS/MS method showed acceptable analytical performance in quantifying PEth as a sensitive and specific biomarker for evaluating alcohol consumption. Results from this study may provide an opportunity to educate women of reproductive age on drinking during pregnancy and the long-term effects of alcohol use.
    Keywords:  PEth ratio; PLPEth; POPEth; alcohol biomarker; positivity rate; pregnancy; women of reproductive age
    DOI:  https://doi.org/10.1093/jat/bkae100
  46. Molecules. 2024 Dec 24. pii: 2. [Epub ahead of print]30(1):
    Protein Precipitation by Metal Hydroxides as a Convenient and Alternative Sample Preparation Procedure for Bioanalysis.
    Emanuele Salina, Luca Regazzoni.
      Protein precipitation is widely used for sample preparation ahead of liquid chromatography. This step is required to analyze small molecules without the interference of proteins contained in the matrix. Organic solvents and acidic chemicals are the two most popular reagents used for this scope. Organic solvents are quite effective precipitating agents, but require a medium-to-large sample dilution. Moreover, a high concentration of organic solvents in sample media can affect reversed phase separations. Therefore, an evaporation step, followed by the resuspension of the analytes in appropriate media, is sometimes required. On the contrary, the addition of acidic compounds is more straightforward, since it keeps the supernatant aqueous and does not require evaporation, but the extreme pH can cause the degradation of analytes and the stationary phase. Herein, an alternative method for protein precipitation using the addition of zinc hydroxide was tested. The main advantages of this method over the other precipitating reagents are the minimal sample dilution required and the maintenance of aqueous media at nearly neutral pH which ensure analyte stability. The protocol ensured an effective protein removal before the analysis of small molecules in biological matrices, resulting in full compatibility with reversed phase chromatography coupled with both UV and mass spectrometric detectors.
    Keywords:  UV spectroscopy; liquid chromatography; mass spectrometry; metal hydroxides; protein precipitation
    DOI:  https://doi.org/10.3390/molecules30010002
  47. Methods Mol Biol. 2025 ;2891 205-219
    A Protocol for GC-MS Profiling of Chiral Secondary Amino Acids.
    Stanislav Opekar, Helena Zahradníčková, Petr Vodrážka, Lucie Řimnáčová, Martin Moos, Petr Šimek.
      A simple analytical workflow is described for gas chromatographic-mass spectrometric (GC-MS)-based chiral profiling of secondary amino acids (AAs) in biological matrices. The sample preparation is carried out directly in aqueous biological sample extracts and involves in situ heptafluorobutyl chloroformate (HFBCF) derivatization-liquid-liquid microextraction of nonpolar products into hexane phase followed by subsequent formation of the corresponding methylamides from the HFB esters by direct treatment with methylamine reagent solution. The (O, N) HFB-butoxycarbonyl-methylamide AA products (HFBOC-MA) are separated on a Chirasil-L-Val capillary column and quantitatively measured by GC-MS operated in selected ion monitoring (SIM) mode. The protocol includes 12 simple pipetting steps and covers the quantitative analysis of 8 L, D pairs of secondary amino acids, including proline, isomeric 3-, 4-hydroxyprolines, pipecolic acid, nipecotic acid, azetidine-2-carboxylic acid, and cis- and trans-5-hydroxy-L-pipecolic acid using 13C5 -L-proline as an internal standard. The individual analytical steps are commented on and explained, with emphasis on the chiral GC-MS analysis of secondary amino acids in human urine, serum, and peptide hydrolysate samples.
    Keywords:  Alkyl chloroformate derivatization; Amino acids; Chiral analysis; GC-MS; Peptide hydrolysates; Quantitative analysis; Secondary amino acids; Serum; Urine
    DOI:  https://doi.org/10.1007/978-1-0716-4334-1_11
  48. J Mass Spectrom. 2025 Feb;60(2): e5112
    Feasibility of IR-MALDESI Mass Spectrometry Imaging of PFAS.
    Allen Martin, Alena N Joignant, Matt Farrell, Antonio Planchart, David C Muddiman.
      Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a class of emerging contaminants that have been in use industrially since the 1940s. Their long-term and extensive commercial use has led to their ubiquitous presence in the environment. The ability to measure the bioconcentration and distribution of PFAS in the tissue of aquatic organisms helps elucidate the persistence of PFAS as well as environmental impacts. Traditional analysis by LC-MS/MS can measure total PFAS concentrations within an organism but cannot provide comprehensive spatial information regarding PFAS concentrations within the organism. In the current study, we used infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) to determine the limit of detection (LOD) of several PFAS utilizing a commercial standard mix spotted on mouse liver tissue. The traditional ice matrix and an alternative matrix, 1,8-bis (tetramethylguanidino)naphthalene (TMGN), were explored when determining the limits of detection for various PFAS by IR-MALDESI. The ice matrix alone resulted in a higher response than the combination of TMGN and ice. The resulting LOD for perfluorooctane sulfonic acid (PFOS) on a per voxel basis was 0.16 fmol/voxel. For comparison, zebrafish that were exposed to perfluorooctanoic acid (PFOA), PFOS, and perfluorohexanesulfonic acid (PFHxS) at different concentrations were homogenized, and PFAS were extracted by solid-liquid extraction, purified by solid phase extraction, and analyzed by LC-MS/MS to determine the level of bioaccumulation in the zebrafish. PFOS resulted in the highest level of bioaccumulation (731.9 μg/kg, or 234.2 fg/voxel). A zebrafish that had been exposed to a PFAS mixture of PFOA (250 ng/L), PFOS (250 ng/L), and PFHxS (125 ng/L) was cryosectioned and analyzed by IR-MALDESI. Images could not be generated as the accumulation of PFAS in the sectioned tissue was below detection limit of the technique.
    Keywords:  IR‐MALDESI; LC–MS/MS; PFAS; mass spectrometry imaging; zebrafish
    DOI:  https://doi.org/10.1002/jms.5112
  49. Animals (Basel). 2024 Dec 24. pii: 3. [Epub ahead of print]15(1):
    Development and Validation of a Robust and Straightforward LC-MS Method for Measuring Taurine in Whole Blood and Plasma of Dogs and Reference Intervals Calculation.
    Tommaso Furlanello, Riccardo Masti, Francesca Maria Bertolini, Valeria Ongaro, Andrea Zoia, Jose Sanchez Del Pulgar.
      Several studies have highlighted the essential role of taurine in maintaining the health of small animals, particularly dogs. Taurine deficiency has been linked to various health issues, especially in certain dog breeds. Therefore, accurately assessing taurine levels in canine blood is crucial for diagnosing and monitoring these conditions. In this study, we present the development of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for rapidly quantifying taurine concentrations in dog whole blood and plasma. The method was validated according to current guidelines, showing excellent accuracy, precision, and sensitivity across a wide concentration range. Specifically, the limit of quantification was set at 80 nmol/mL for whole blood and 8 nmol/mL for plasma, ensuring the method's reliability for both matrices. The application of this validated technique to blood samples of healthy dogs allowed for the establishment of reference intervals for taurine concentrations (148 to 495 nmol/mL for whole blood; 42 to 183 nmol/mL for plasma). Due to its robustness and simplicity, this method represents a valuable tool, supporting its routine use in health assessments and enabling more effective monitoring of taurine status in dogs.
    Keywords:  LC-MS/MS; method validation; reference interval; taurine
    DOI:  https://doi.org/10.3390/ani15010003
  50. J Chromatogr Sci. 2025 Jan 14. pii: bmaf005. [Epub ahead of print]63(2):
    Method Validation for Estimation of Imidacloprid and its Metabolites in Maize and Soil by LCMS-MS.
    Sandeep Kaur, Smriti Sharma, Balpreet K Kang.
      Validation of Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method was performed for estimation of imidacloprid (IM) and its metabolites in maize leaves, immature kernels, mature kernels, stalk, and soil using liquid chromatograph tandem mass spectrometry, coupled with electrospray ionization. The extraction in different matrices of maize and soil was performed using acetonitrile +0.1% formic acid followed by clean-up with primary secondary amine sorbent and anhydrous magnesium sulfate. The method was validated in terms of selectivity, linearity, limit of detection, limit of quantification, matrix effect, ion ratios, quality control, robustness, accuracy, and precision. The validation of all parameters was done in accordance with European Commission's Directorate-General for Health and Food Safety (DG SANTE) guidelines. A linear relationship with high correlation coefficients R2 > 0.99 was obtained for solvents and different matrices viz., maize leaves, immature kernels, mature kernels, stalk, and soil. The recovery and relative standard deviations were ˃78% and ˂5.4%, respectively. This method permits a simple, sensitive, accurate, cost-effective, precise, and rapid extraction of IM and its metabolites from maize leaves, immature kernels, mature kernels, stalks, and soil. This can help the residue analysts to address effective residue estimation, regular monitoring of residues and can also aid in the regulatory and food safety concerns about the usage of IM in maize.
    Keywords:  Imidacloprid; LCMS-MS; Maize; Method validation; QuEChERS
    DOI:  https://doi.org/10.1093/chromsci/bmaf005
  51. J Am Soc Mass Spectrom. 2025 Jan 13.
    New Library-Based Methods for Nontargeted Compound Identification by GC-EI-MS.
    Deborah F McGlynn, Lindsay D Yee, H Martin Garraffo, Lewis Y Geer, Tytus D Mak, Yuri A Mirokhin, Dmitrii V Tchekhovskoi, Coty N Jen, Allen H Goldstein, Anthony J Kearsley, Stephen E Stein.
      While gas chromatography mass spectrometry (GC-MS) has long been used to identify compounds in complex mixtures, this process is often subjective and time-consuming and leaves a large fraction of seemingly good-quality spectra unidentified. In this work, we describe a set of new mass spectral library-based methods to assist compound identification in complex mixtures. These methods employ mass spectral uniqueness and compound ubiquity of library entries alongside noise reduction and automated comparison of retention indices to library compounds. As a test data set, we used a publicly available electron ionization mass spectrometry data set consisting of 4833 spectra of particulate organic compounds emitted by combustion of wildland fuels. In the present work, spectra in this data set were first identified using the NIST 2023 EI-MS Library and associated batch process identification software (NIST MS PepSearch) using retention-index corrected Identity Search scoring. Resulting identifications and related information were then employed to parametrize other factors that correlate with identification. A method for identifying compounds absent from but related to those present in mass spectral libraries using the Hybrid Similarity Search is illustrated. Nevertheless, some 90% of the spectra remain unidentified. Through comparison of unidentified to identified mass spectra in this data set, a new simple measure, namely median relative abundance, was developed for evaluating the likelihood of identification.
    DOI:  https://doi.org/10.1021/jasms.4c00451
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