bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2024–10–06
fifty papers selected by
Sofia Costa, Matterworks
  1. Global Metabolomics Using LC-MS for Clinical Applications.
  2. Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.
  3. Recommendations for Accurate Lipid Annotation and Semi-absolute Quantification from LC-MS/MS Datasets.
  4. Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.
  5. Distinguishing Artifactual Fatty Acid Dimers from Fatty Acid Esters of Hydroxy Fatty Acids in Untargeted LC-MS Pipelines.
  6. Modular, Scalable, and Customizable LC-HRMS for Exposomics.
  7. High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids.
  8. Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.
  9. Quantification of Octadecanoids in Human Plasma Using Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry.
  10. LC-MS/MS Quantification of Endocannabinoids in Tissues.
  11. CE-MS-Based Clinical Metabolomics of Human Plasma.
  12. Buffer-free high pH mobile phase LC-MS/MS for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 and 20 drugs and metabolites in whole blood.
  13. Rapid Lipid Mediator Profiling by Convergence Chromatography-MS/MS.
  14. Prostaglandin Metabolites Analysis in Urine by LC-MS/MS.
  15. Analysis of the Mammalian Lipidome by DMS Shotgun Lipidomics.
  16. Development of a Simple and Validated LC-MS/MS Method for Quantitative Determination of Ketotifen in Beagle Dog Plasma and Its Application to Bioequivalence Study of Ketotifen Syrup Dosage Form.
  17. Development of an advanced analytical technique for detecting multiple pesticide residues in vegetables through liquid chromatography tandem mass spectroscopy (LC-MS/MS).
  18. Determination of ivermectin in plasma and whole blood using LC-MS/MS.
  19. (Pre)Clinical Metabolomics Analysis.
  20. Combined LC-MS/MS feature grouping, statistical prioritization, and interactive networking in msFeaST.
  21. Development and Validation of an LC-MS/MS Assay for the Quantitation of MO-OH-Nap Tropolone in Mouse Plasma: Application to In Vitro and In Vivo Pharmacokinetic Studies.
  22. Faster liquid chromatography-tandem mass spectrometry method for analysis of isomeric urinary mercapturic acid metabolites of crotonaldehyde, methacrolein, and methyl vinyl ketone.
  23. Advances in Chiral Metabolomic Profiling and Biomarker Discovery.
  24. Extracting Knowledge from MS Clinical Metabolomic Data: Processing and Analysis Strategies.
  25. Annotation of Metabolites in Stable Isotope Tracing Untargeted Metabolomics via Khipu-web.
  26. Optimized high-throughput protocols for comprehensive metabolomic and lipidomic profiling of brain sample.
  27. Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.
  28. Stable Isotope Tracing Experiments Using LC-MS.
  29. Rapid Analysis of Sterols in Blood-Derived Samples.
  30. A Comprehensive Spatially Resolved Metabolomics Dataset for Lampreys.
  31. A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.
  32. Development of Simultaneous Drug Concentration Measurement Method Using an Automated Pretreatment Liquid Chromatography/Tandem Mass Spectrometry System for Therapeutic Drug Monitoring.
  33. NMR-Based Analysis of Cellular Central Energy Metabolism and Exchange Rates.
  34. Long Chain Base Profiling with Multiple Reaction Monitoring Mass Spectrometry.
  35. Fast determination of organophosphate esters and their metabolites in human matrices by a straightforward cold-induced strategy coupled with HPLC-MS/MS.
  36. NMR-Based Stable Isotope Tracing of Cancer Metabolism.
  37. Mass Spectrometry Advancements and Applications for Biomarker Discovery, Diagnostic Innovations, and Personalized Medicine.
  38. Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.
  39. In Situ Isotope Tracing at Single-Cell Resolution Using Mass Spectrometry Imaging.
  40. Chemical Derivatization and Paper Spray Ionization Mass Spectrometry for Fast Screening of Retinoic Acid in Cosmetics.
  41. Lipoprotein (Sub)Fraction Analysis on the Bruker B.I. LISA Platform.
  42. Nucleo-SAFARI: Automated Identification of Fragment Ions in Top-Down MS/MS Spectra of Nucleic Acids.
  43. Pharmacokinetic study of granisetron in human plasma measured by UPLC-MS/MS and its use in healthy Chinese subjects.
  44. An LCMS/MS method for the simultaneous determination of ten antimicrobials and its application in critically ill patients.
  45. Untargeted Metabolomics for Inborn Errors of Metabolism: Development and Evaluation of a Sustainable Reference Material for Correcting Inter-Batch Variability.
  46. Development and validation of multiresidue analysis method for biomonitoring of pesticides and metabolites in human blood and urine by LC-QToF-MS.
  47. Differential Mobility Spectrometry-Based Cardiolipin Analysis.
  48. Exploring natural product biosynthesis in plants with mass spectrometry imaging.
  49. A comparative UPLC-orbitrap-MS-based metabolite profiling of three Pelargonium species cultivated in Egypt.
  50. Liquid-Liquid Extraction Solvent Selection for Comparing Illegal Drugs in Whole Blood and Dried Blood Spot with LC-MS/MS.
  1. Methods Mol Biol. 2025 ;2855 23-39
    Global Metabolomics Using LC-MS for Clinical Applications.
    Hanne Bendiksen Skogvold, Elise Sandås Sand, Katja Benedikte Prestø Elgstøen.
      Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.
    Keywords:  Clinical metabolomics; Data quality evaluation; Dried blood spots; Global metabolomics; LC-MS; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_2
  2. Methods Mol Biol. 2025 ;2855 185-194
    Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.
    Zuzana Vaňková, Ondřej Peterka, Robert Jirásko, Michal Holčapek.
      Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 μm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.
    Keywords:  High-throughput lipidomics; Mass spectrometry; Plasma; Quantitation; Reversed-phase; Serum; Ultrahigh-performance liquid chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_12
  3. Methods Mol Biol. 2025 ;2855 269-287
    Recommendations for Accurate Lipid Annotation and Semi-absolute Quantification from LC-MS/MS Datasets.
    Michele Wölk, Maria Fedorova.
      Recent developments in LC-MS instrumentation and analytical technologies together with bioinformatics tools supporting high-throughput processing of large omics datasets significantly enhanced our capabilities and efficiency of identification and quantification of lipids in diverse biological materials. However, each biological matrix is characterized by its unique lipid composition, thus requiring optimization of analytical and bioinformatics workflows for each studied lipidome. Here, we describe an integrated workflow for deep lipidome profiling, accurate annotation, and semi-absolute quantification of complex lipidomes based on reversed phase chromatography and high resolution mass spectrometry. This chapter provides details on selection of the optimal extraction protocol, acquisition of LC-MS/MS data for accurate annotation of lipid molecular species, and design of lipidome-specific mixtures of internal standards to assist quantitative analysis of complex, native lipidomes.
    Keywords:  High resolution mass spectrometry; Lipid annotations; Lipidomics; Quantitative lipidomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_16
  4. Methods Mol Biol. 2025 ;2855 305-314
    Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.
    Ondřej Peterka, Denise Wolrab, Robert Jirásko, Mária Kanásová, Zuzana Dolečková, Michal Holčapek.
      Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.
    Keywords:  High-throughput lipidomics; Mass spectrometry; Plasma; Quantitation; Serum; Ultrahigh-performance supercritical fluid chromatography; Validation
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_18
  5. Methods Mol Biol. 2025 ;2855 67-84
    Distinguishing Artifactual Fatty Acid Dimers from Fatty Acid Esters of Hydroxy Fatty Acids in Untargeted LC-MS Pipelines.
    Alisa B Nelson, Eric D Queathem, Patrycja Puchalska.
      Untargeted metabolomics is a powerful profiling tool for the discovery of possible biomarkers of disease onset and progression. Analytical pipelines applying liquid chromatography (LC) and mass spectrometry (MS)-based methods are widely used to survey a broad range of metabolites within various metabolic pathways, including organic acids, amino acids, nucleosides, and lipids. Accurate and complete identification of putative metabolites is an ongoing challenge in untargeted metabolomics studies. Highly sensitive instrumentation can result in the detection of adduct and fragment ions that form reproducibly and contain identifiable ions that are difficult to distinguish from metabolic pathway intermediates, which may result in false-positive identification. At concentrations as low as 10 μM, free fatty acids have been found to form homo- and heterodimers in untargeted metabolomics pipelines that resemble the lipid class fatty acid esters of hydroxy fatty acids (FAHFAs), resulting in misidentification. This chapter details a protocol for LC-MS-based untargeted metabolomics using hydrophilic interaction chromatography (HILIC) that specifically aids in distinguishing artifactual fatty acid dimers from endogenous FAHFAs.
    Keywords:  Fatty acid dimers; Fatty acid esters of hydroxy fatty acids; LC-MS; Lipidomics; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_4
  6. Methods Mol Biol. 2025 ;2855 41-66
    Modular, Scalable, and Customizable LC-HRMS for Exposomics.
    Vinicius Verri Hernandes, Benedikt Warth.
      In this chapter, we describe a multi-purpose, reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflow for acquiring high-quality, non-targeted exposomics data utilizing data-dependent acquisition (DDA) combined with the use of toxicant inclusion lists for semi-targeted analysis. In addition, we describe expected retention times for >160 highly diverse xenobiotics in human plasma and serum samples. The method described is intended to serve as a generic LC-HRMS exposomics workflow for research and educational purposes. Moreover, it may be employed as a primer, allowing for further adaptations according to specialized research needs, e.g., by including reference and/or internal standards, by expanding to data-independent acquisition (DIA), or by modifying the list of compounds prioritized in fragmentation experiments (MS2).
    Keywords:  Analytical toxicology; Biomonitoring; Data-dependent analysis; Environmental and food contaminants; Exposome research; High-resolution mass spectrometry; Untargeted metabolomics and exposomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_3
  7. Methods Mol Biol. 2025 ;2855 195-207
    High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids.
    Stefan Schauer, Alaa Othman.
      Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1-12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5 min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1-500 μM is achieved for most SCFAs, while it is 0.05-100 μM for MCFAs. Validation of the procedure depicts good linearity (R2 > 0.98) and repeatability (CV ≤ 20%). The lower limit of detection (LLOD) is 10-30 nM. The lower limit of quantification (LLOQ) is 50-100 nM for most analytes, while it is 0.5 μM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.
    Keywords:  3-Nitrophenylhydrazine derivatization; Branched-short-chain fatty acids; Cancer therapy; Gut microbiome; Immune system; Medium-chain fatty acids; Personalized medicine; Short-chain fatty acids; Signaling molecules; Targeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_13
  8. Methods Mol Biol. 2025 ;2855 117-131
    Quantification of Total Ketone Bodies in Biological Matrices Using Stable Isotope-Labeled Internal Standards and RP-UHPLC-MS/MS.
    Eric D Queathem, Alisa B Nelson, Patrycja Puchalska.
      Acetoacetate (AcAc) and D-beta-hydroxybutyrate (D-βOHB), the two major ketone bodies found in circulation, are linked to multiple physiological and pathophysiological states. Therefore, analytical methodologies surrounding the quantification of total ketone body (TKB) concentrations in biological matrices are paramount. Traditional methods to quantify TKBs relied on indirect spectrophotometric assays with narrow dynamic ranges, which have been significantly improved upon by modern mass spectrometry (MS)-based approaches. However, the lack of stable isotope-labeled internal standards (ISs) for AcAc and the need to distinguish D-βOHB from its closely related structural and enantiomeric isomers pose significant obstacles. Here, we provide a protocol to synthesize and quantify a [13C] stable isotope-labeled IS for AcAc, which, in conjunction with a commercially available [2H] stable isotope-labeled IS for βOHB, allows TKBs to be measured across multiple biological matrices. This rapid (7 min) analysis employs reverse phase ultra-high performance liquid chromatography (RP-UHPLC) coupled to tandem MS (MS/MS) to distinguish βOHB from three structural isomers using parallel reaction monitoring (PRM), providing excellent specificity and selectivity. Finally, a method is provided that distinguishes D-βOHB from L-βOHB using a simple one-step derivatization to produce the corresponding diastereomers, which can be chromatographically resolved using the same rapid RP-UHPLC separation with new PRM transitions. In summary, this method provides a rigorous analytical pipeline for the analysis of TKBs in biological matrices via leveraging two authentic stable isotope-labeled ISs and RP-UHPLC-MS/MS.
    Keywords:  Acetoacetate; D-β-hydroxybutyrate; Ketone bodies; L-β-hydroxybutyrate; Stable isotope-labeled internal standards; Tandem mass spectrometry; Ultra-high performance liquid chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_7
  9. Methods Mol Biol. 2025 ;2855 315-339
    Quantification of Octadecanoids in Human Plasma Using Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry.
    Olivier Salamin, Craig E Wheelock.
      Octadecanoids are a subset of oxylipins derived from 18-carbon fatty acids. These compounds have historically been understudied but have more recently attracted attention to their purported biological activity. One obstacle to the study of octadecanoids has been a lack of specific analytical methods for their measurement. A particular limitation has been the need for chiral-based methods that enable separation and quantification of individual stereoisomers. The use of chirality provides an additional dimension for distinguishing analytes produced enzymatically from those formed through autoxidation. In this chapter, we describe a comprehensive method using chiral supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) for the quantification of octadecanoids in human plasma. This method stands as an effective approach for quantifying octadecanoids and is applicable to diverse research applications including clinical research.
    Keywords:  Chiral separation; Lipid mediators; Mass spectrometry; Octadecanoids; Supercritical fluid chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_19
  10. Methods Mol Biol. 2025 ;2855 133-145
    LC-MS/MS Quantification of Endocannabinoids in Tissues.
    Anna Hoekstra, Martin Giera, Elena Sánchez-López.
      Endocannabinoids (ECBs) are lipid-derived endogenous molecules with important physiological roles such as regulation of energy balance, immunity, or neural development. Quantitation of ECBs helps better understand their physiological role and modulation of biological processes. This chapter presents the simultaneous quantification of 14 ECBs and related molecules in the brain, liver, and muscle, as well as white and brown adipose tissue using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The dynamic range of the method has been tuned to cover the endogenous concentrations of these analytes given the fact that they are endogenously present at different orders of magnitude. Specifically, three groups are established: 0.5-5000 ng/mL for 2-oleoyl- and 2-linoleoylglycerol and arachidonic acid, 0.05-500 ng/mL for 2-arachidonoylglycerol, and 0.0005-0.5 ng/mL for anandamide, palmitoyl-, palmitoleoyl-, stearoyl-, oleoyl-, linoleoyl-, alpha-linolenoyl-, dihomo-gamma-linolenoyl-, docosahexaenoyl-, and pentadecanoylethanolamide.
    Keywords:  Brain; Brown adipose tissue; Endocannabinoids; LC-MS/MS; Liver; Muscle; White adipose tissue
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_8
  11. Methods Mol Biol. 2025 ;2855 389-423
    CE-MS-Based Clinical Metabolomics of Human Plasma.
    Maricruz Mamani-Huanca, Sara Martínez, Ángeles López-López, Ángeles López-Gonzálvez, Oihane E Albóniga, Ana Gradillas, Coral Barbas, Víctor González-Ruiz.
      Capillary electrophoresis coupled to mass spectrometry (CE-MS) has emerged as a powerful analytical technique with significant implications for clinical research and diagnostics. The integration of information from CE and MS strengthens confidence in the identification of compounds present in clinical samples. The ability of CE to separate molecules based on their electrophoretic mobility coupled to MS enables the accurate identification and quantification of analytes, even in complex biological matrices such as human plasma.Here, we present a detailed protocol for an untargeted metabolomics study using CE-MS and its application in a study on human plasma from patients suffering Long COVID syndrome. The protocol ranges from sample preparation to biological interpretation, detailing a workflow enabling the analysis of cationic and anionic compounds, metabolite identification, and data processing.
    Keywords:  CE-MS; Clinical metabolomics; Human plasma; Long COVID; Polar metabolome; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_23
  12. Talanta. 2024 Sep 27. pii: S0039-9140(24)01343-2. [Epub ahead of print]282 126964
    Buffer-free high pH mobile phase LC-MS/MS for determination of the alcohol biomarker phosphatidylethanol 16:0/18:1 and 20 drugs and metabolites in whole blood.
    Benedicte Jørgenrud, Tao McQuade, Marisa H Maria, Galina Nilsson, Thomas Berg.
       BACKGROUND: Acidic mobile phases are commonly used in reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) bioanalysis. However, increased sensitivity, improved peak symmetry, and increased retention, especially for basic hydrophilic drugs have been observed using basic mobile phases. In our previous acidic mobile phase LC-MS/MS method we needed two injections (0.4 and 2.0 μL) of each sample for this task, which is inefficient. The aim of this study was to investigate if basic mobile phase LC-MS/MS could be used to determine phosphatidylethanol 16:0/18:1 and 20 other drugs and metabolites with satisfactory sensitivity in one single run.
    METHODS: Whole blood was prepared by 96-well supported-liquid extraction using heptane/ethyl acetate/2-propanol (16:64:20, v:v:v). Chromatographic separation was achieved on an Acquity BEH C18 column (50 × 2.1 mm I.D.), using a mobile phase with 0.025 % ammonia, pH 10.7 (Solvent A) and methanol (Solvent B). All compounds had isotope-labelled internal standards.
    RESULTS: The method was fully validated. Recovery was between 63 and 91 % for 20 compounds and 10 % for benzoylecgonine. Matrix effects were low, except for ion enhancement of buprenorphine and ion suppression for THC. However, internal standards compensated for these effects. Inter-assay precision and accuracy were < ± 20 % for all compounds at five tested concentrations, except for methamphetamine at the highest concentration.
    CONCLUSION: An LC-MS/MS method for simultaneous determination of PEth 16:0/18:1 and 20 drugs and metabolites in whole blood were for the first time developed and validated. Retention of PEth 16:0/18:1 was, in contrast to the other 20 compounds, largely affected by mobile phase buffer concentration. The buffer free basic mobile phase ensured that phosphatidylethanol 16:0/18:1 eluted before most of the unwanted phospholipids.
    Keywords:  96-Well supported liquid extraction; Basic mobile phase; Drugs; LC-MS/MS; Phosphatidylethanol 16:0/18:1; Whole blood
    DOI:  https://doi.org/10.1016/j.talanta.2024.126964
  13. Methods Mol Biol. 2025 ;2855 341-354
    Rapid Lipid Mediator Profiling by Convergence Chromatography-MS/MS.
    Guille Metzler, Richard C King, Carmen Fernández-Metzler, Amitava Das, T Gregg Davis, Besnik Bajrami, Tom Bretschneider, David Kvaskoff.
      Bioactive lipid mediators derived from arachidonic acid constitute an attractive pool of metabolites that reflect cellular function and signaling, as well as potential biomarkers that may respond quantitatively to disease progression or pharmacological treatment. Their quantitative measurement in biological samples is complicated by the number of isomers that share common structural features, which are not easily distinguished by immunoassays or reverse phase chromatography-tandem mass spectrometry. Here, we present a method that enables the rapid analysis of a panel of over 25 biologically important eicosanoids in a 96-well format for cell culture supernatants, plasma, and organ tissues using convergence chromatography-tandem mass spectrometry to resolve these analytes of interest.
    Keywords:  Convergence chromatography; High-throughput; Oxylipins; Sample pre-treatment
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_20
  14. Methods Mol Biol. 2025 ;2855 147-154
    Prostaglandin Metabolites Analysis in Urine by LC-MS/MS.
    Guille Metzler, Lily A Day, Richard C King, Carmen Fernández-Metzler, Amitava Das, T Gregg Davis, Besnik Bajrami, Tom Bretschneider, David Kvaskoff.
      The analysis of prostaglandin urinary metabolites is valuable for assessing physiological processes and identifying disease biomarkers. These metabolites, derived from the breakdown of prostaglandins, offer a noninvasive means to gauge prostaglandin production and its potential impact on various biological functions. We report an efficient LC-MS method of four commonly analyzed prostaglandin urinary metabolites including tetranor-PGEM (derived from PGE2), tetranor-PGDM, 11β-PGF2α, and 2,3-dinor-11β-PGF2α (derived from PGD2). Each metabolite possesses distinct characteristics and clinical applications, collectively contributing to our understanding of prostaglandin-mediated pathways.
    Keywords:  APCI; Assay robustness; Prostaglandins; Urinary metabolites
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_9
  15. Methods Mol Biol. 2025 ;2855 357-372
    Analysis of the Mammalian Lipidome by DMS Shotgun Lipidomics.
    Baolong Su, Kevin J Williams.
      Shotgun Lipidomics is a robust methodology for the characterization of the lipidome of complex biological samples. This assay is among the most quantitative lipidomics methods and is capable of surveying a wide breadth of lipid subclasses, both neutral and polar. The shortfalls of the technique include limitations in lipid species characterization and computationally demanding data analysis requiring isotopic and isobaric overlap correction. Differential Mobility Spectrometry (DMS) has demonstrated its utility in enabling acyl tail characterization within a Shotgun Lipidomics experiment. Here, we present a workflow for DMS Shotgun Lipidomics that measures 1400 possible lipid species. It utilizes the Shotgun Lipidomics Assistant (SLA) application, an open-source application that supervises the data analysis for an expansive Shotgun Lipidomics experiment.
    Keywords:  DMS; Direct infusion; Mammalian lipidome; Shotgun lipidomics; Shotgun lipidomics assistant
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_21
  16. Molecules. 2024 Sep 23. pii: 4505. [Epub ahead of print]29(18):
    Development of a Simple and Validated LC-MS/MS Method for Quantitative Determination of Ketotifen in Beagle Dog Plasma and Its Application to Bioequivalence Study of Ketotifen Syrup Dosage Form.
    Eunseo Song, Wang-Seob Shim, Doowon Choi, Yuna Song, Hyeong Geun Jo, Soobok Lee, Suk Han Jung, Yeo Jin Choi, Kyung-Tae Lee.
      A highly accurate, precise, and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for ketotifen (KTF) estimation from Beagle dog plasma was developed and validated, with ketotifen-d3 (KTF-d3) as the internal standard (IS). KTF and IS were detected on an API 4000 mass spectrometer in multiple reaction monitoring (MRM) mode in electrospray ionization (ESI) positive ionization mode. The transitions were monitored at m/z 310.2 → 96.0 for KTF and m/z 313.2 → 99.1 for IS. KTF and IS were extracted from plasma using liquid-liquid extraction with methyl tertiary-butyl ether and then analyzed for 3 min with extracted samples (7 µL) into the LC-MS/MS system. Analytes were separated on a Luna® Hilic column (50 × 2.0 mm i.d., 3 μm) using the Nexera X2 HPLC. The mobile phase A consisted of 10 mmol/L ammonium formate (pH 3.0), while mobile phase B consisted of 0.05% formic acid in acetonitrile. The ratio of mobile phase was 5:95 (v/v) at a flow rate of 0.2 mL/min. The method has been thoroughly validated in accordance with the bioanalytical method validation guidelines established by the Ministry of Food and Drug Safety in Korea and the U.S. Food and Drug Administration, addressing selectivity, lower limit of quantification, linearity, carryover, precision, accuracy, recovery, matrix effect, and stability. The developed LC-MS/MS method was effectively utilized for the bioequivalence assessment of ketotifen in Beagle dog plasma following the oral administration of ketotifen syrup.
    Keywords:  Beagle dog plasma; LC–MS/MS; bioanalytical method validation; bioequivalence; ketotifen; syrup
    DOI:  https://doi.org/10.3390/molecules29184505
  17. J Environ Sci Health B. 2024 ;59(10): 663-677
    Development of an advanced analytical technique for detecting multiple pesticide residues in vegetables through liquid chromatography tandem mass spectroscopy (LC-MS/MS).
    Sujan Majumder, Arvind Kumar, Sadhan Debnath, Abhinay, A N Singh, T K Behera.
      A comprehensive LC-MS/MS method, which employs Positive Electrospray Ionization (PEI) and Multiple Reaction Monitoring (MRM) was developed for the simultaneous determination of 35 pesticides belonging to various chemical classes in tomato, brinjal, chili, and okra samples. Extraction was facilitated using a modified QuEChERS method, which allows efficient sample analysis in a single run. Calibration curves for each pesticide exhibited linearity within the concentration range of 0.0025 to 0.1 µg mL-1, with correlation coefficients ranging from 0.993 to 0.999. Mean recoveries at five fortification levels (0.01 to 0.5 µg kg-1) ranged from 80 to 90%, demonstrating satisfactory precision (RSD < 20%). The matrix effects, mitigated through an optimized cleanup process, were observed within the range of 6.42% to 19.52%. The developed method having the limit of quantification of 0.01 mg kg-1 for all 35 pesticides, proved to be highly sensitive and rapid for multi-residue estimation in diverse vegetable samples. Subsequently, the method was used to analyze the market samples from Varanasi, India, which revealed the presence of pesticides like Chlorpyrifos, Chlorantraniliproleand Indoxacarb in tomato, brinjal, chili and okra. Therefore, the method could be considered as a robust tool for monitoring pesticide residues in vegetables, aiding in quality assessment and regulatory compliance in the agriculture sector.
    Keywords:  LC-MS/MS; Method optimization; market samples; multiple residues; vegetables
    DOI:  https://doi.org/10.1080/03601234.2024.2407713
  18. Wellcome Open Res. 2024 ;9 231
    Determination of ivermectin in plasma and whole blood using LC-MS/MS.
    Natpapat Kaewkhao, Warunee Hanpithakpong, Joel Tarning, Daniel Blessborn.
       Background: Ivermectin is a widely used drug for the treatment of helminthiasis and filariasis worldwide, and it has also shown promise for malaria elimination through its potent mosquito-lethal activity. The objective of this study was to develop and validate a high-throughput and sensitive method to quantify ivermectin in plasma and whole blood samples, using automated sample extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
    Methods: Phospholipids were removed in patient whole blood (100 µl) and plasma (100 µl) samples using a 96-well plate Hybrid-solid phase extraction technique. Ivermectin and its isotope-labelled internal standard (ivermectin-D2) were separated on an Agilent Poroshell 120 EC-C18 50mm × 3.0mm I.D. 2.7µm, using a mobile phase of acetonitrile: ammonium formate 2 mM containing 0.5% formic acid (90: 10, v/v). Detection was performed using a triple quadrupole mass spectrometer in the positive ionization mode.
    Results: The method was validated in the concentration range 0.970 - 384 ng/ml in both plasma and whole blood matrices. Intra- and inter-batch precisions during the validation were below 15%. There was no carryover or matrix effects detected. Ivermectin is a stable compound and results showed no degradation in the different stability tests.
    Conclusions: The validated method proved to have high sensitivity and precision, good selectivity and to be suitable for clinical application or laboratory quantification of ivermectin in plasma or whole blood samples.
    Keywords:  Development; Human blood; Ivermectin; LC-MS/MS; Malaria; Validation
    DOI:  https://doi.org/10.12688/wellcomeopenres.20613.2
  19. Methods Mol Biol. 2025 ;2855 3-19
    (Pre)Clinical Metabolomics Analysis.
    Ratna Budhi Pebriana, Elena Sánchez-López, Martin Giera.
      Metabolomics is the scientific field with the eager goal to comprehensively analyze the entirety of all small molecules of a biological system, i.e., the metabolome. Over the last few years, metabolomics has matured to become an analytical cornerstone of life science research across diverse fields, from fundamental biochemical applications to preclinical studies, including biomarker discovery and drug development. In this chapter, we provide an introduction to (pre)clinical metabolomics. We define key metabolomics aspects and provide the basis to thoroughly understand the relevance of this field in a biological and clinical context. We present and explain state-of-the-art analytical technologies devoted to metabolomic analysis as well as emerging technologies, discussing both strengths and weaknesses. Given the ever-increasing demand for handling complex datasets, the role of bioinformatics approaches in the context of metabolomic analysis is also illustrated.
    Keywords:  Clinical samples; Lipidomics; Mass spectrometry; Metabolomics; Nuclear magnetic resonance
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_1
  20. Bioinformatics. 2024 Sep 30. pii: btae584. [Epub ahead of print]
    Combined LC-MS/MS feature grouping, statistical prioritization, and interactive networking in msFeaST.
    Kevin Mildau, Christoph Büschl, Jürgen Zanghellini, Justin J J van der Hooft.
       SUMMARY: Computational metabolomics workflows have revolutionized the untargeted metabolomics field. However, the organization and prioritization of metabolite features remains a laborious process. Organizing metabolomics data is often done through mass fragmentation-based spectral similarity grouping, resulting in feature sets that also represent an intuitive and scientifically meaningful first stage of analysis in untargeted metabolomics. Exploiting such feature sets, feature-set testing has emerged as an approach that is widely used in genomics and targeted metabolomics pathway enrichment analyses. It allows for formally combining groupings with statistical testing into more meaningful pathway enrichment conclusions. Here, we present msFeaST (mass spectral Feature Set Testing), a feature-set testing and visualization workflow for LC-MS/MS untargeted metabolomics data. Feature-set testing involves statistically assessing differential abundance patterns for groups of features across experimental conditions. We developed msFeaST to make use of spectral similarity-based feature groupings generated using k-medoids clustering, where the resulting clusters serve as a proxy for grouping structurally similar features with potential biosynthesis pathway relationships. Spectral clustering done in this way allows for feature group-wise statistical testing using the globaltest package, which provides high power to detect small concordant effects via joint modeling and reduced multiplicity adjustment penalties. Hence, msFeaST provides interactive integration of the semi-quantitative experimental information with mass-spectral structural similarity information, enhancing the prioritization of features and feature sets during exploratory data analysis.
    AVAILABILITY AND IMPLEMENTATION: The msFeaST workflow is freely available through https://github.com/kevinmildau/msFeaST and built to work on MacOS and Linux systems.
    SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.
    Keywords:  data visualization; mass spectral grouping; molecular networking; statistical testing; untargeted metabolomics
    DOI:  https://doi.org/10.1093/bioinformatics/btae584
  21. Molecules. 2024 Sep 18. pii: 4424. [Epub ahead of print]29(18):
    Development and Validation of an LC-MS/MS Assay for the Quantitation of MO-OH-Nap Tropolone in Mouse Plasma: Application to In Vitro and In Vivo Pharmacokinetic Studies.
    Wafaa N Aldhafiri, Yashpal S Chhonker, Nusrat Ahmed, Sandeep K Singh, Staci L Haney, James B Ford, Sarah A Holstein, Daryl J Murry.
      A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of MO-OH-Nap tropolone (MO-OH-Nap) in mouse plasma. MO-OH-Nap is an α-substituted tropolone with anti-proliferative properties in various cancer cell lines. Detection and separation of analytes was achieved on an ACE Excel C18 (1.7 µm, 100 × 2.1 mm, MAC-MOD Analytical, Chadds Ford, PA, USA) column with mobile phase consisting of 0.05% trifluoroacetic acid in water (mobile phase A) and 0.05% trifluoroacetic acid in acetonitrile (mobile phase B), with an isocratic elution of 15:85% (A:B) at a total flow rate of 0.25 mL/min. The LC-MS/MS system was operated at unit resolution in multiple reaction monitoring (MRM) mode, using precursor ion > product ion combination of 249.10 > 202.15 m/z for MO-OH-Nap and 305.10 > 215.05 m/z for the internal standard (IS), BA-SM-OM. The MS/MS response was linear over a concentration range of 1 to 500 ng/mL with a correlation coefficient (r2) of ≥0.987. The within- and between-batch precision (%RSD) and accuracy (%Bias) were within acceptable limits. The validated method was successfully applied to determine MO-OH-Nap metabolic stability, plasma protein binding, and bio-distribution studies of MO-OH-Nap in CD-1 mice.
    Keywords:  LC-MS/MS; biodistribution; in vitro metabolism; plasma protein binding; tropolone
    DOI:  https://doi.org/10.3390/molecules29184424
  22. J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 19. pii: S1570-0232(24)00333-7. [Epub ahead of print]1247 124324
    Faster liquid chromatography-tandem mass spectrometry method for analysis of isomeric urinary mercapturic acid metabolites of crotonaldehyde, methacrolein, and methyl vinyl ketone.
    Menglan Chen, Steven G Carmella, Yingchun Zhao, Stephen S Hecht.
      We report a significantly more rapid method for quantitation of the urinary mercapturic acids of the isomeric toxicants crotonaldehyde, methacrolein, and methyl vinyl ketone. The major innovation of this novel method is detection by liquid chromatography-negative atmospheric pressure chemical ionization-tandem mass spectrometry-selected reaction monitoring as opposed to detection by negative electrospray ionization in our previously reported method. The new method also uses an improved Raptor Biphenyl HPLC column. The total chromatographic analysis time was reduced to about 8 min compared to 35 min in our previously published method. Accuracy, precision, and ruggedness of the new method were established, and its suitability for the analysis of urine samples from 2500 cigarette smokers and non-smokers was confirmed. The improved method is practical for quantitation of these important toxicants in clinical studies requiring analysis of thousands of urine samples.
    Keywords:  Crotonaldehyde; Liquid chromatography; Methacrolein; Methyl vinyl ketone; Tandem mass spectrometry; Urinary mercapturic acids
    DOI:  https://doi.org/10.1016/j.jchromb.2024.124324
  23. Methods Mol Biol. 2025 ;2855 85-101
    Advances in Chiral Metabolomic Profiling and Biomarker Discovery.
    Renu Pandey, Stefano Tiziani.
      Chiral metabolomics entails the enantioselective measurement of the metabolome present in a biological system. Over recent years, it has garnered significant interest for its potential in discovering disease biomarkers and aiding clinical diagnostics. D-Amino acids and D-hydroxy acids, traditionally overlooked as unnatural, are now emerging as novel signaling molecules and potential biomarkers for a range of metabolic disorders, brain diseases, kidney disease, diabetes, and cancer. Despite their significance, simultaneous measurements of multiple classes of chiral metabolites in a biological system remain challenging. Hence, limited information is available regarding the metabolic pathways responsible for synthesizing D-amino/hydroxy acid and their associated pathophysiological mechanisms in various diseases. Capitalizing on recent advancements in sensitive analytical techniques, researchers have developed various targeted chiral metabolomic methods for the analysis of chiral biomarkers. Here, we highlight the pivotal role of chiral metabolic profiling studies in disease diagnosis, prognosis, and therapeutic interventions. Furthermore, we describe cutting-edge chromatographic and mass spectrometry methods that enable enantioselective analysis of chiral metabolites. These advanced techniques are instrumental in unraveling the complexities of disease biomarkers, contributing to the ongoing efforts in disease biomarker discovery.
    Keywords:  Biomarker discovery; Chiral metabolomics; D-amino/hydroxy acids; Enantioselective measurement
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_5
  24. Methods Mol Biol. 2025 ;2855 539-554
    Extracting Knowledge from MS Clinical Metabolomic Data: Processing and Analysis Strategies.
    Isabel Meister, Julien Boccard, Serge Rudaz.
      Assessing potential alterations of metabolic pathways using large-scale approaches plays today a central role in clinical research. Because several thousands of mass features can be measured for each sample with separation techniques hyphenated to mass spectrometry (MS) detection, adapted strategies have to be implemented to detect altered pathways and help to elucidate the mechanisms of pathologies. These procedures include peak detection, sample alignment, normalization, statistical analysis, and metabolite annotation. Interestingly, considerable advances have been made over the last years in terms of analytics, bioinformatics, and chemometrics to help massive and complex metabolomic data to be more adequately handled with automated processing and data analysis workflows. Recent developments and remaining challenges related to MS signal processing, metabolite annotation, and biomarker discovery based on statistical models are illustrated in this chapter in light of their application to clinical research.
    Keywords:  Data analysis, Annotation; Data processing; Mass spectrometry; Metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_29
  25. J Am Soc Mass Spectrom. 2024 Sep 30.
    Annotation of Metabolites in Stable Isotope Tracing Untargeted Metabolomics via Khipu-web.
    Joshua M Mitchell, Yuanye Chi, Shujian Zheng, Maheshwor Thapa, Eric Wang, Shuzhao Li.
      Stable isotope tracing is a crucial technique for understanding the metabolic wiring of biological systems, determining metabolic flux through pathways of interest, and detecting novel metabolites and pathways. Despite the potential insights provided by this technique, its application remains limited to a small number of targeted molecules and pathways. Because previous software tools usually require chemical formulas to find relevant features, and the data are highly complex, especially in untargeted metabolomics and when the downstream reactions and metabolites are poorly characterized. We report here Khipu version 2 and its new user-friendly web application. New functions are added to enhance analyzing stable isotope tracing data including metrics that evaluate peak enrichment in labeled samples, scoring methods to facilitate robust detection of intensity patterns and integrated natural abundance correction. We demonstrate that this approach can be applied to untargeted metabolomics to systematically extract isotope-labeled compounds and annotate the unidentified metabolites.
    DOI:  https://doi.org/10.1021/jasms.4c00175
  26. Talanta. 2024 Sep 26. pii: S0039-9140(24)01332-8. [Epub ahead of print]282 126953
    Optimized high-throughput protocols for comprehensive metabolomic and lipidomic profiling of brain sample.
    Cemil Can Eylem, Emirhan Nemutlu, Aysegul Dogan, Vedat Acik, Selcuk Matyar, Yurdal Gezercan, Suleyman Altintas, Ali Ihsan Okten, Nursabah Elif Basci Akduman.
      Establishing direct causal and functional links between genotype and phenotype requires thoroughly analyzing metabolites and lipids in systems biology. Tissue samples, which provide localized and direct information and contain unique compounds, play a significant role in objectively classifying diseases, predicting prognosis, and deciding personalized therapeutic strategies. Comprehensive metabolomic and lipidomic analyses in tissue samples need efficient sample preparation steps, optimized analysis conditions, and the integration of orthogonal analytical platforms because of the physicochemical diversities of biomolecules. Here, we propose simple, rapid, and robust high-throughput analytical protocols based on the design of experiment (DoE) strategies, with the various parameters systematically tested for comprehensively analyzing the heterogeneous brain samples. The suggested protocols present a systematically DoE-based strategy for performing the most comprehensive analysis for integrated GC-MS and LC-qTOF-MS from brain samples. The five different DoE models, including D-optimal, full factorial, fractional, and Box-Behnken, were applied to increase extraction efficiency for metabolites and lipids and optimize instrumental parameters, including sample preparation and chromatographic parameters. The superior simultaneous extraction of metabolites and lipids from brain samples was achieved by the methanol-water-dichloromethane (2:1:3, v/v/v) mixture. For GC-MS based metabolomics analysis, incubation time, temperature, and methoxyamine concentration (10 mg/mL) affected metabolite coverage significantly. For LC-qTOF-MS based metabolomics analysis, the extraction solvent (methanol-water; 2:1, v/v) and the reconstitution solvent (%0.1 FA in acetonitrile) were superior on the metabolite coverage. On the other hand, the ionic strength and column temperature were critical and significant parameters for high throughput metabolomics and lipidomics studies using LC-qTOF-MS. In conclusion, DoE-based optimization strategies for a three-in-one single-step extraction enabled rapid, comprehensive, high-throughput, and simultaneous analysis of metabolites, lipids, and even proteins from a 10 mg brain sample. Under optimized conditions, 475 lipids and 158 metabolites were identified in brain samples.
    Keywords:  Brain; Experimental design; Lipidomics; Metabolomics; Multi-omics; Sample preparation
    DOI:  https://doi.org/10.1016/j.talanta.2024.126953
  27. J Mass Spectrom. 2024 Oct;59(10): e5088
    Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.
    Arpon Biswas, Abhijit Deb Choudhury, Anjali Mishra, Sarvesh Kumar Verma, Amol Chhatrapati Bisen, Sachin Nashik Sanap, Sristi Agrawal, Mukesh Kumar, Shivansh Kumar, Rabi Sankar Bhatta.
      Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.
    Keywords:  LC–MS/MS; antibiotic; corticosteroid; eye drop; tear fluid
    DOI:  https://doi.org/10.1002/jms.5088
  28. Methods Mol Biol. 2025 ;2855 103-116
    Stable Isotope Tracing Experiments Using LC-MS.
    Esther A Zaal, Jeroen W A Jansen, Celia R Berkers.
      Metabolomics has emerged as a pivotal field in understanding cellular function, particularly in the context of disease. In numerous diseases, including cancer, alterations in metabolism play an essential role in disease progression and drug response. Hence, unraveling the metabolic rewiring is of importance to find novel diagnostic and therapeutic strategies. Isotope tracing is a powerful technique for delving deeper into the metabolic wiring of cells. By tracking an isotopically labeled substrate through biochemical reactions in the cell, this technique provides a dynamic understanding of cellular metabolism. This chapter outlines a robust isotope tracing protocol utilizing high-resolution mass spectrometry coupled to liquid chromatography in cell culture-based models. We cover essential aspects of experimental design and analyses, providing a valuable resource for researchers aiming to employ isotopic tracing.
    Keywords:  Fluxomics; High-performance liquid chromatography; Isotope tracing; Mass spectrometry; Metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_6
  29. Methods Mol Biol. 2025 ;2855 291-302
    Rapid Analysis of Sterols in Blood-Derived Samples.
    Ratna Budhi Pebriana, Selma Simsek, Marieke Heijink, Martin Giera.
      Dysregulations of cholesterol biosynthesis are known to be associated with several pathologies. Due to the rapid growth of clinical investigations in this research area, a specific, fast, and valid method for analyzing cholesterol, its precursors, and metabolites is required. Here, we describe a rapid method for sample preparation, separation, and quantification of sterols in blood-derived samples using polymeric solid phase extraction followed by gas chromatography-mass spectrometry. The validated method demonstrates a reliable quantification of cholesterol, its precursors, and metabolites.
    Keywords:  Cholesterol; Gas chromatography-mass spectrometry; Hydroxycholesterol; Targeted metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_17
  30. Sci Data. 2024 Oct 02. 11(1): 1075
    A Comprehensive Spatially Resolved Metabolomics Dataset for Lampreys.
    Meng Gou, Xiaxia Wang, Xuyuan Duan, Yaocen Wang, Yue Pang, Yonghui Dong.
      As one of the two most ancient groups of extant vertebrates, lamprey has become an important model organism in various fields of biology. In this paper, we present a comprehensive tissue-wide spatial metabolomics dataset for lampreys, where 14 distinct tissues were analyzed using liquid chromatography-mass spectrometry (LC-MS) in both positive and negative ion modes. The dataset has been fully validated using internal standard and pooled quality control samples and is readily accessible at the UCSD Metabolomics Workbench. This dataset serves as a valuable resource for researchers using lampreys as a model organism. Additionally, it acts as a benchmark metabolomics dataset for evaluating new algorithms and software tools and comparing them with previously published results. A lamprey spatial metabolomics database is also provided to support studies utilizing lampreys as an animal model, and to complement and validate other spatial metabolomics studies on lampreys conducted with mass spectrometry imaging or other techniques.
    DOI:  https://doi.org/10.1038/s41597-024-03925-6
  31. Methods Mol Biol. 2025 ;2855 155-169
    A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.
    Mohan Ghorasaini, Daniela Costa, Victoria J Tyrrell, Majd Protty, Martin Giera, Valerie B O'Donnell.
      Oxidized phospholipids (oxPLs) are generated during innate immunity and inflammation, where they play a variety of biological roles, including regulation of autoimmunity and coagulation. Some are generated by enzymatic reactions, leading to stereo- and regiospecificity, while many others can be formed through nonenzymatic oxidation and truncation and can be used as biomarkers of oxidative stress. Mass spectrometry methods have been developed over many years for oxPL analysis, which can provide robust estimations of molecular species and amounts, where standards are available. Here we present a method used for the analysis of enzymatically-generated oxPL (eoxPL), which allows quantification of mono-hydroxy oxylipin-containing species. We also show profiling of many other partially characterized structures in tissue samples and provide typical chromatograms obtained.
    Keywords:  Inflammatory mediators; Liquid chromatography; Mass spectrometry; Oxidized phospholipids; Oxylipins
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_10
  32. Pharmaceutics. 2024 Aug 28. pii: 1138. [Epub ahead of print]16(9):
    Development of Simultaneous Drug Concentration Measurement Method Using an Automated Pretreatment Liquid Chromatography/Tandem Mass Spectrometry System for Therapeutic Drug Monitoring.
    Yu Sato, Hiroki Kondo, Yuji Sato, Ai Abe, Masafumi Kikuchi, Toshihiro Sato, Masaki Kumondai, Kohei Yoshikawa, Yoshihiro Hayakawa, Masamitsu Maekawa, Nariyasu Mano.
      Therapeutic drug monitoring (TDM) is a personalized treatment approach that involves optimizing drug dosages based on patient-specific factors, such as drug plasma concentrations, therapeutic efficacy, or adverse reactions. The plasma concentration of drugs is determined using liquid chromatography/tandem mass spectrometry (LC-MS/MS) or various immunoassays. Compared with immunoassays, LC-MS/MS requires more pretreatment time as the number of samples increases. Recently, fully automated pretreatment LC-MS/MS systems have been developed to automatically perform whole-sample pretreatment for LC-MS/MS analysis. In this study, we developed a method for simultaneous concentration determination of five analytes (clozapine, mycophenolic acid, sunitinib, N-desethylsunitinib, and voriconazole) using LC-MS/MS for clinical TDM using a fully automated LC-MS/MS pretreatment system. In the developed method, the intra- and inter-assay relative error (RE) values ranged between -14.8% and 11.3%; the intra- and inter-assay coefficient of variation (CV) values were <8.8% and <10.5%, respectively. The analytes showed good stability, with RE values ranging between -13.6% and 10.9% and CV values <8.9%. Furthermore, the plasma concentrations in clinical samples using this method and the conventional manual pretreatment method showed similar results. Therefore, the method developed in this study could be considered a useful pretreatment method for routine TDM in patients.
    Keywords:  LC-MS/MS; automated sample preparation; plasma concentration; simultaneous measurement method of drug efficacy; therapeutic drug monitoring
    DOI:  https://doi.org/10.3390/pharmaceutics16091138
  33. Methods Mol Biol. 2025 ;2855 505-519
    NMR-Based Analysis of Cellular Central Energy Metabolism and Exchange Rates.
    Sarantos Kostidis.
      Cell cultures are widely used in studies to gain mechanistic insights of metabolic processes. The foundation of these studies lies on the quantification of intracellular and extracellular metabolites, and nuclear magnetic resonance (NMR) is one of the key analytical platforms used to this aim. Among the factors influencing the quality of the produced data are the sampling procedures as well as the acquisition and processing of spectroscopic data. Here we provide our workflow for obtaining quantitative metabolic data from adherent mammalian cells using NMR spectroscopy. The described protocol is compatible with other analytical methods like LC- or GC-MS-based lipidomics and untargeted metabolomics from the same sample. We also show how the collected extracellular data can be used to extract exchange flux rates, particularly useful for flux analysis studies and metabolic engineering of human-induced pluripotent stem cells.
    Keywords:  Central energy metabolism; Exchange flux rates; Mammalian cells; Metabolic engineering; Metabolomics; NMR spectroscopy; Sample preparation
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_27
  34. Methods Mol Biol. 2025 ;2855 209-223
    Long Chain Base Profiling with Multiple Reaction Monitoring Mass Spectrometry.
    Andreas J Hülsmeier, Lavanya Gunasegaram, Florine Wipfli, Museer A Lone, Thorsten Hornemann.
      Sphingolipids (SLs) are essential lipids with important functions in membrane formation and cell signaling. The presence of a long chain base (LCB) structure is common to all SLs. De novo SL synthesis is initiated by the enzyme serine-palmitoyltransferase (SPT), which forms an LCB by the conjugation from serine and fatty acyl-CoAs. SPT can metabolize a variety of acyl-CoA substrates, which form diverse LCB structures within and across species. The LCB then undergoes further metabolic modifications resulting in an extraordinarily diverse spectrum of sphingolipids formed. SL analysis, using liquid chromatography-mass spectrometry (LC-MS)-based methods, poses challenges due to the diverse range of frequently isobaric species. This complexity complicates the identification of underlying LCB structures using standard lipidomics approaches. Here, we describe a simplified method to analyze the LCB profile in cells, tissue, and blood. The procedure involves chemical hydrolysis to remove the conjugated headgroups and N-acyl chains, allowing to specifically resolve the underlying LCB structures by LC-MS. This method can also be combined with an isotope labeling approach to determine in vivo SPT activity and total SL de novo synthesis over time.
    Keywords:  Liquid chromatography mass spectrometry; Long chain base; Multiple reaction monitoring; Serine-palmitoyltransferase; Sphingolipids
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_14
  35. Anal Bioanal Chem. 2024 Oct 05.
    Fast determination of organophosphate esters and their metabolites in human matrices by a straightforward cold-induced strategy coupled with HPLC-MS/MS.
    Jiaxin Wu, Shunying Yao, Yiwei Wang, Sai Fan, Xia Cui, Dawei Chen, Zhixiong Shi.
      Organophosphate esters (OPEs) are commonly used chemicals and are also regarded as emerging environmental pollutants. Recently, it has been proved that metabolites of OPEs (mOPEs) could also cause health concerns. However, analytical methods for the concurrent measurement of OPEs and mOPEs in human matrices are still complicated. In this study, a convenient and efficient analytical method combining a cold-induced strategy and HPLC-MS/MS was developed to simultaneously determine 18 OPEs and 10 mOPEs in human serum, urine, and human milk. In brief, after the sample was extracted with acetonitrile, a "one-step" treatment combining purification and enrichment was accomplished by cold-induced liquid-liquid extraction (CI-LLE), and analytes were then quantified by HPLC-ESI-MS/MS. The ratio of acetonitrile/water, and the temperature and time set in the CI-LLE procedure were optimized for achieving the highest enrichment factors. Under the best conditions, linearity, limits of detection (LODs), recovery, precision, and matrix effects of OPEs/mOPEs were verified. LODs of OPEs/mOPEs in serum, urine, and human milk were 0.1-113 pg/mL, 0.1-22 pg/mL, and 0.2-22 pg/mL, respectively. Average recoveries ranged from 80 to 123%, with relative standard deviations lower than 15% for most analytes. The matrix effect test showed slight signal enhancement or inhibition, and the use of isotopically labeled internal standards (ISs) could compensate for the effects. In real sample analysis, both OPEs and mOPEs showed high detecting frequency, which indicated their ubiquity in humans.
    Keywords:  Cold-induced extraction; Human milk; Organophosphate esters; Serum; Urine
    DOI:  https://doi.org/10.1007/s00216-024-05572-7
  36. Methods Mol Biol. 2025 ;2855 457-504
    NMR-Based Stable Isotope Tracing of Cancer Metabolism.
    Penghui Lin, Andrew N Lane, Teresa W-M Fan.
      NMR is widely used for metabolite profiling (metabolomics, metabonomics) particularly of various readily obtainable biofluids such as plasma and urine. It is especially valuable for stable isotope tracer studies to track metabolic pathways under control or perturbed conditions in a wide range of cell models as well as animal models and human subjects. NMR has unique properties for utilizing stable isotopes to edit or simplify otherwise complex spectra acquired in vitro and in vivo, while quantifying the level of enrichment at specific atomic positions in various metabolites (i.e., isotopomer distribution analysis).In this protocol, we give an overview with specific protocols for NMR-based stable isotope-resolved metabolomics, or SIRM, with a workflow from administration of isotope-enriched precursors, via sample preparation through to NMR data collection and reduction. We focus on indirect detection of common NMR-active stable isotopes including 13C, 15N, 31P, and 2H, using a variety of 1H-based two-dimensional experiments. We also include the application and analyses of multiplex tracer experiments.
    Keywords:  Isotopomer distribution analysis; NMRNuclear magnetic resonance (NMR); Spectral editing; Stable isotope-resolved metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_26
  37. Int J Mol Sci. 2024 Sep 12. pii: 9880. [Epub ahead of print]25(18):
    Mass Spectrometry Advancements and Applications for Biomarker Discovery, Diagnostic Innovations, and Personalized Medicine.
    Ahrum Son, Woojin Kim, Jongham Park, Yongho Park, Wonseok Lee, Sangwoon Lee, Hyunsoo Kim.
      Mass spectrometry (MS) has revolutionized clinical chemistry, offering unparalleled capabilities for biomolecule analysis. This review explores the growing significance of mass spectrometry (MS), particularly when coupled with liquid chromatography (LC), in identifying disease biomarkers and quantifying biomolecules for diagnostic and prognostic purposes. The unique advantages of MS in accurately identifying and quantifying diverse molecules have positioned it as a cornerstone in personalized-medicine advancement. MS-based technologies have transformed precision medicine, enabling a comprehensive understanding of disease mechanisms and patient-specific treatment responses. LC-MS has shown exceptional utility in analyzing complex biological matrices, while high-resolution MS has expanded analytical capabilities, allowing the detection of low-abundance molecules and the elucidation of complex biological pathways. The integration of MS with other techniques, such as ion mobility spectrometry, has opened new avenues for biomarker discovery and validation. As we progress toward precision medicine, MS-based technologies will be crucial in addressing the challenges of individualized patient care, driving innovations in disease diagnosis, prognosis, and treatment strategies.
    Keywords:  biomarker discovery; clinical chemistry; liquid chromatography–mass spectrometry; personalized medicine
    DOI:  https://doi.org/10.3390/ijms25189880
  38. Methods Mol Biol. 2025 ;2855 171-183
    Nonenzymatic Oxidized Polyunsaturated Fatty Acid Products in Human Plasma and Urine Samples by LC-QTOF-MS/MS.
    Kin Sum Leung, Jetty Chung-Yung Lee.
      Oxidative stress induces autooxidation of polyunsaturated fatty acids, producing numerous isoprostanoids and isofuranoids. These oxidized products are measurable in human plasma and urine and serve as oxidative stress biomarkers for chronic diseases. This chapter details the preparation and measurement of α-linolenic acid-derived phytoprostanes and phytofurans in human samples using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS/MS).
    Keywords:  Liquid chromatography; PUFA oxidation products; Phytofurans; Phytoprostanes; Tandem mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_11
  39. Methods Mol Biol. 2025 ;2855 523-535
    In Situ Isotope Tracing at Single-Cell Resolution Using Mass Spectrometry Imaging.
    Rosalie G J Rietjens, Bram Heijs.
      Mass spectrometry imaging (MSI) allows for label-free spatial molecular interrogation of tissues. With advances in the field over recent years, the spatial resolution at which MSI data can be recorded has reached the single-cell level. This makes MSI complementary to other single-cell omics technologies. As metabolism is a highly dynamic process, capturing the metabolic turnover adds a valuable layer of information. Here, we describe how to set up in situ stable isotope tracing followed by MSI-enabled spatial metabolomics to perform dynamic metabolomics at the single-cell level.
    Keywords:  Isotope tracing; Mass spectrometry imaging; Tissue metabolomics; Vibratome sectioning
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_28
  40. Molecules. 2024 Sep 21. pii: 4491. [Epub ahead of print]29(18):
    Chemical Derivatization and Paper Spray Ionization Mass Spectrometry for Fast Screening of Retinoic Acid in Cosmetics.
    Yuzhang Bao, Ningzi Guo, Xiaowen Hu, Bin Di, Yang Liu, Huimin Sun.
      As a prescription drug, retinoic acid is listed as a banned cosmetic additive in the EU and China regulations. Currently, spectrophotometric methods, including thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and HPLC-MS/MS, are commonly used for the determination of retinoic acid. As these conventional methods require complex pretreatment and are time-consuming, chemical derivatization combined with paper spray ionization mass spectrometry was developed for the fast detection of retinoic acid in cosmetics. N,N-dimethylpiperazine iodide (DMPI) was utilized as a derivatization reagent. Carboxylic acid in retinoic acid was derivatized to carry a positive charge and was subjected to mass spectrometry analysis. Results showed that compared with non-derivatized compounds, the detection limit was increased by about 50 times. The linearity in the range of 0.005-1 μg·mL-1 was good. The limit of detection (LOD) was 0.0013 μg·mL-1, and the limit of quantification (LOQ) was 0.0043 μg·mL-1. The recoveries of spiked samples were in the range of 95-105%, and the RSDs were below 5%. Derivatization and paper spray ionization MS render a quick, sensitive, and accurate method for the detection of retinoic acid in a complex matrix.
    Keywords:  PSI-MS; chemical derivatization; cosmetics; retinoic acid
    DOI:  https://doi.org/10.3390/molecules29184491
  41. Methods Mol Biol. 2025 ;2855 445-456
    Lipoprotein (Sub)Fraction Analysis on the Bruker B.I. LISA Platform.
    Aswin Verhoeven.
      The Bruker B.I. LISA platform provides a method for human plasma/serum lipoprotein analysis and yields data on the particle numbers and lipids of the main lipoprotein classes (VLDL, IDL, LDL, HDL), and the subfractions within those classes. In order to obtain quantitative and reproducible results, the prescribed protocol, the B.I. Methods, needs to be followed. In this chapter, the B.I. Methods protocol steps relevant for B.I. LISA analyses are described.
    Keywords:  Lipid classes; Lipoprotein classes; Lipoproteins; NMR; Plasma; Serum; Subfractions
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_25
  42. Anal Chem. 2024 Oct 04.
    Nucleo-SAFARI: Automated Identification of Fragment Ions in Top-Down MS/MS Spectra of Nucleic Acids.
    Michael B Lanzillotti, Jennifer S Brodbelt.
      Recent progress in top-down mass spectrometry analysis of progressively larger nucleic acids has enabled in-depth characterization of intact, modified RNA molecules. Development of methods for desalting and MS/MS fragmentation allows rapid acquisition of high-quality top-down MS/MS spectra of nucleic acids up to 100 nt, which has spurred the need for development of software approaches to identify and validate nucleic acid fragment ions. We have implemented an R-based approach to aid in analysis of MS/MS spectra of nucleic acids based on fragment ions observed directly in the m/z domain. This program, entitled Shiny Application for Fragment Assignment by Relative Isotopes (Nucleo-SAFARI), utilizes the Shiny HTML framework for deployment of a user-friendly application for automated annotation of top-down MS/MS spectra of nucleic acids recorded on Orbitrap mass spectrometer platforms. This approach proceeds through in silico generation of fragment ions and their isotopic distributions, followed by algorithmic assessment of the experimental isotopic distributions. Nucleo-SAFARI is available for download at https://github.com/mblanzillotti/Nucleo-SAFARI.
    DOI:  https://doi.org/10.1021/acs.analchem.4c03201
  43. Pak J Pharm Sci. 2024 May;37(3): 475-489
    Pharmacokinetic study of granisetron in human plasma measured by UPLC-MS/MS and its use in healthy Chinese subjects.
    Lingying Jiang, Lei Mao, Jie Lin, Jiangmin Cheng, Zebei Liu, Hangyu Zhao, Xin Zhang, Chenfang Wang, Linfang Deng.
      In this study, a rapid, simple and sensitive UPLC-MS/MS method was established for the quantification of granisetron in human plasma for the prevention of vomiting after radiotherapy and chemotherapy. The precipitated proteins were extracted and gradient eluted on a ZORBAX Eclipse Plus C18 column (2.1×50mm, 1.8μm) to achieve ideal chromatographic separation. Multiple reaction mode (MRM) was performed using a Turboion Spray API5500 mass spectrometer equipped with Electron Spray Ionization (ESI). For method validation, good linearity was observed for each analyte of interest in the validation concentration range of 0.05 to 20.0ng/mL. The CV% of inter-batch and intra-batch precision were in the range of -3.6% to 4.7% and the precision of both inter-batch and intra-batch was ≤15.0%. In addition, the method had the advantage of a low matrix effect. In human plasma, all analytes remained stable for 2 hours when kept at room temperature; samples were stable within the autosampler (5°C) for 141 h after preparation and after four freeze-thaw cycles at -20°C and -70°C for 48 days. The UPLC-MS method that had been validated was later utilized for the pharmacokinetic investigation of granisetron hydrochloride tablets in orally administered doses to healthy Chinese volunteers, both before and after meals.
  44. J Pharm Biomed Anal. 2024 Sep 30. pii: S0731-7085(24)00531-4. [Epub ahead of print]252 116489
    An LCMS/MS method for the simultaneous determination of ten antimicrobials and its application in critically ill patients.
    Tingting Liu, Xiaomin Chen, Guanxuanzi Zhang, Jing Zhao, Qian Lu, Fang Wang, Hongxia Li, Bing Liu, Ping Zhu.
      Significant pharmacokinetic variation occurs in critically ill patients, leading to underexposure to antibiotics and poor prognosis. In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LCMS/MS) platform for the simultaneous quantification of 8 antibacterial and 2 antifungal drugs, which is optimally suited for clinically efficient, real-time therapeutic drug monitoring (TDM). Multiple reaction monitoring (MRM) mass spectrometry was used in this method, and samples were prepared via protein precipitation with methanol. Chromatographic separation was accomplished on a BGIU Column-U02 (2.1ｘ50 mm, 3 µm), with six stable isotopes and one analog as an internal standard. The overall turnaround time of the assay was 5 minutes. All the drugs tested (piperacillin, cefoperazone, meropenem, levofloxacin, moxifloxacin, daptomycin, linezolid, vancomycin, fluconazole and voriconazole) were linear in the test concentration range (r ≥ 0.9900), the accuracy was 95 %-111 %, the precision variation coefficient was greater than or equal to 10 %, the lower limit of quantitation was 0.31-7.51 mg/L, and the coefficient of variation of the matrix factor was less than 10 %. The recovery rates ranged from 85 % to 115 %, and the antibiotics were stable at 4°C and -20°C for 6 days, with an offset of greater than or equal to 15 %. This method was successfully applied to routine TDM in 252 elderly critically ill patients.
    Keywords:  Antibiotics; Critically ill patients; LC‒MS/MS method; Pharmacokinetics/pharmacodynamics; Therapeutic drug monitoring
    DOI:  https://doi.org/10.1016/j.jpba.2024.116489
  45. Clin Chem. 2024 Oct 04. pii: hvae141. [Epub ahead of print]
    Untargeted Metabolomics for Inborn Errors of Metabolism: Development and Evaluation of a Sustainable Reference Material for Correcting Inter-Batch Variability.
    Rafael Garrett, Adam S Ptolemy, Sara Pickett, Mark D Kellogg, Roy W A Peake.
       BACKGROUND: Untargeted metabolomics has shown promise in expanding screening and diagnostic capabilities for inborn errors of metabolism (IEMs). However, inter-batch variability remains a major barrier to its implementation in the clinical laboratory, despite attempts to address this through normalization techniques. We have developed a sustainable, matrix-matched reference material (RM) using the iterative batch averaging method (IBAT) to correct inter-batch variability in liquid chromatography-high-resolution mass spectrometry-based untargeted metabolomics for IEM screening.
    METHODS: The RM was created using pooled batches of remnant plasma specimens. The batch size, number of batch iterations per RM, and stability compared to a conventional pool of specimens were determined. The effectiveness of the RM for correcting inter-batch variability in routine screening was evaluated using plasma collected from a cohort of phenylketonuria (PKU) patients.
    RESULTS: The RM exhibited lower metabolite variability between iterations over time compared to metabolites from individual batches or individual specimens used for its creation. In addition, the mean variation across amino acid (n = 19) concentrations over 12 weeks was lower for the RM (CVtotal = 8.8%; range 4.7%-25.3%) compared to the specimen pool (CVtotal = 24.6%; range 9.0%-108.3%). When utilized in IEM screening, RM normalization minimized unwanted inter-batch variation and enabled the correct classification of 30 PKU patients analyzed 1 month apart from 146 non-PKU controls.
    CONCLUSIONS: Our RM minimizes inter-batch variability in untargeted metabolomics and demonstrated its potential for routine IEM screening in a cohort of PKU patients. It provides a practical and sustainable solution for data normalization in untargeted metabolomics for clinical laboratories.
    DOI:  https://doi.org/10.1093/clinchem/hvae141
  46. Talanta. 2024 Sep 27. pii: S0039-9140(24)01347-X. [Epub ahead of print]282 126968
    Development and validation of multiresidue analysis method for biomonitoring of pesticides and metabolites in human blood and urine by LC-QToF-MS.
    Vaibhavi Lahane, Sakshi Singh, Sachin Mishra, Akhilesh K Yadav.
      The widespread use of pesticides and their consequential presence in the environment is a growing concern due to the harmful health effects associated with pesticide exposure. For clinical and toxicology laboratories, a method for simultaneously determining these compounds and their metabolic products in body fluids, such as blood and urine, is important. In the present study, a rapid, sensitive and simultaneous LC-QToF-MS method for detecting multiclass pesticides and metabolites in blood and urine samples has been developed and validated. Four sample preparation procedures, protein precipitation and three different variants of QuEChERS-based extraction were evaluated to find a suitable, simple, and effective sample pretreatment technique. The final optimized sample preparation method (acetonitrile; 400 μl, MgSO4; 40 mg and NaCl; 10 mg) was validated for accuracy, precision, matrix effect, recovery, stability, carryover, and dilution integrity. Analyte recoveries ranged from 75.40 to 113.54 % while accuracy was evaluated in the range of 71.41-108.26 % and precision (%RSD) in the range of 0.01 %-16.85 %. The limit of quantification (LOQ) for all compounds was established in the range of 0.82-7.05 ng mL-1. The developed reliable, robust, and sensitive method was successfully applied for the quantification of target pesticides and metabolites in human blood and urine samples. Evaluated samples resulted in detection of eleven analytes (seven pesticides and four metabolites).
    Keywords:  Biomonitoring; Metabolites; Pesticide residues; QToF-MS; QuEChERS
    DOI:  https://doi.org/10.1016/j.talanta.2024.126968
  47. Methods Mol Biol. 2025 ;2855 373-385
    Differential Mobility Spectrometry-Based Cardiolipin Analysis.
    Fabien Riols, Michael Witting, Mark Haid.
      Cardiolipins (CL) are special lipids in many respects. First of all, CL are composed of four fatty acids linked by two phosphatidic acids, which provide CL a unique molecular structure. Secondly, in eukaryotic cells they are specific to a single organelle, mitochondria, where they are also synthetized. CL are one of the most abundant lipid classes in mitochondria, mainly localized in the inner membrane. They are key determinants of mitochondrial health and homeostasis by modulating membrane integrity and fluidity, mitochondrial shapes, and metabolic pathways. Disturbances in mitochondrial CL composition can lead to tissue malfunction and diseases. It is therefore important to develop analytical tools to study the mitochondrial lipidome, and more particularly the CL. The method described here allows the quantification of cardiolipins at the sum composition level in isolated mitochondria or in liver tissue by flow injection analysis coupled to differential mobility spectrometry (FIA-DMS), also known as DMS-based shotgun lipidomics.
    Keywords:  Cardiolipins; Differential mobility spectrometry; Flow injection analysis; Mass spectrometry; Mitochondria
    DOI:  https://doi.org/10.1007/978-1-0716-4116-3_22
  48. Trends Plant Sci. 2024 Sep 27. pii: S1360-1385(24)00231-0. [Epub ahead of print]
    Exploring natural product biosynthesis in plants with mass spectrometry imaging.
    Yuchen Zou, Weiwei Tang, Bin Li.
      The biosynthesis of natural products (NPs) is a complex dynamic spatial and temporal process that requires the collaboration of multiple disciplines to explore the underlying mechanisms. Mass spectrometry imaging (MSI) is a powerful technique for studying NPs due to its high molecular coverage and sensitivity without the need for labeling. To date, many analysts still use MSI primarily for visualizing the distribution of NPs in heterogeneous tissues, although studies have proved that it can provide crucial insights into the specialized spatial metabolic process of NPs. In this review we strive to bring awareness of the importance of MSI, and we advocate further exploitation of the spatial information obtained from MSI to establish metabolite-gene expression relationships.
    Keywords:  biosynthesis; mass spectrometry imaging; multi-omics; natural products; spatial metabolomics
    DOI:  https://doi.org/10.1016/j.tplants.2024.08.002
  49. Sci Rep. 2024 10 01. 14(1): 22765
    A comparative UPLC-orbitrap-MS-based metabolite profiling of three Pelargonium species cultivated in Egypt.
    Rana M Merghany, Mohamed A Salem, Shahira M Ezzat, Sherifa F A Moustafa, Salma A El-Sawi, Meselhy R Meselhy.
      Several Pelargonium species are cultivated mainly to produce essential oils used in perfume industry and for ornamental purposes. Although the chemical composition and biological activities of their essential oils were extensively investigated, there is limited information about the chemical composition of their non-volatile constituents. In this study, we report an Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)-based metabolomics approach for the annotation and analysis of various metabolites in three species; P. graveolens, P. denticulatum, and P. fragrans utilizing The Global Natural Product Social Molecular Networking (GNPS) and multivariate data analyses for clustering of the metabolites. A total of 154 metabolites belonging to different classes were annotated. The three species are good sources of coumarins, benzoic acid derivatives, organic acids, fatty acids, and phospholipids. However, the highest level of flavonols (mono- and di-O-glycosides) and cinnamic acid derivatives was found in P. graveolens and P. denticulatum, whereas tannins and flavone C-glycosides were abundant in P. fragrans. The metabolic profiles clarified here provide comprehensive information on the non-volatile constituents of the three Pelargonium species and can be employed for their authentication and possible therapeutic applications.
    Keywords:   P. denticulatum ; P. fragrans ; P. graveolens ; LC–MS/MS; Metabolomics; Molecular networking
    DOI:  https://doi.org/10.1038/s41598-024-72153-0
  50. J Anal Toxicol. 2024 Oct 05. pii: bkae081. [Epub ahead of print]
    Liquid-Liquid Extraction Solvent Selection for Comparing Illegal Drugs in Whole Blood and Dried Blood Spot with LC-MS/MS.
    Melike Aydoğdu, Hasan Ertaş, Fatma Nil Ertaş, Serap Annette Akgür.
       AIM: This study focused on the simultaneous detection of amphetamine, 3,4-methyl enedioxy methamphetamine, morphine, benzoylecgonine, and 11-nor-9-carboxy- tetrahydrocannabinol (Δ9-THC-COOH) in whole blood and DBS. It is aimed to select a solvent mixture for liquid-liquid extraction (LLE) technique employing LC-MS/MS. The obtained DBS results were compared with the whole blood samples results.
    METHODS: A simple, rapid, and reliable LC-MS/MS method was developed and validated for all analytes in whole blood and DBS. LC was performed on a Hypersil Gold C18 column an initial step with a gradient of 0.01 % formic acid, 5 mM ammonium format buffer in water, and acetonitrile at 0.3 ml/min with 7.5-min runtime.
    RESULTS: A methanol:acetonitrile (40:60 v/v) mixture was selected for both matrices. LOQ values were 10-25 ng/mL; linear ranges were LOQ-500 ng/ml for all analytes; correlation coefficients were greater than 0.99, and all calibrator concentrations were within 20%. Analytical recovery in blood and DBS ranged from 84.9-113.2% of the expected concentration for both intra and-inter day. Analytes were stable for 1, 10, and 30 days after three freeze/thaw cycles. It was determined that the variances of the results obtained with the two matrices in the comparison study were equal for each analyte, and the results were highly correlated (r=0.9625).
    CONCLUSION: A sensitive, accurate, and reliable chromatographic method was developed to determine amphetamine, MDMA, morphine, benzoylecgonine, and cannabis, by performing the same preliminary steps with whole blood and dried blood spots. It was observed that the results obtained in these two matrices were compatible and interchangeable when statistically compared.
    Keywords:  Blood; Dried Blood Spot; LC-MS/MS; Liquid-Liquid Extraction
    DOI:  https://doi.org/10.1093/jat/bkae081
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