bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–08–24
thirteen papers selected by
Sofia Costa, Matterworks



  1. J Mass Spectrom. 2025 Sep;60(9): e5168
      We developed a candidate reference measurement procedure (cRMP) based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the accurate quantification of clozapine (CLO) in human plasma. After systematic optimization of chromatographic separation conditions for CLO, we evaluated various sample pretreatment methods. The analytical performance of the cRMP was rigorously validated, encompassing specificity, recovery, precision, linearity, limit of quantitation (LoQ), limit of detection (LoD), carryover, stability, and method comparison. Measurement uncertainties were assessed in accordance with GUM, taking CLO purity, balance weighing, calibration curve, measurement imprecision, recovery, and carryover into account. Under optimized conditions, CLO showed effective separation from potential interferents in human plasma. The method exhibited excellent linearity (R2 = 0.9988) across a concentration range of 5.65-1693.51 ng/g. The total coefficients of variation (CVs) were 2.04%, 0.97%, and 0.65% at concentrations of 24.53, 98.06, and 987.02 ng/g, respectively. Average recoveries ranged from 97.80% to 99.28%, with a LoQ of 2.73 ng/g and a LoD of 0.91 ng/g. The expanded measurement uncertainties (U) were 1.3 ng/g (k = 2) at 24.53 ng/g, 3.8 ng/g (k = 2) at 98.06 ng/g, and 35.3 ng/g (k = 2) at 987.02 ng/g. The developed cRMP for CLO quantification demonstrates excellent specificity, accuracy, precision, and traceability, meeting all the criteria for cRMPs. This method has the potential to standardize therapeutic drug monitoring (TDM) for CLO, improving clinical outcomes and patient safety.
    Keywords:  ID‐LC–MS/MS; analytical performance; clozapine; measurement uncertainty; therapeutic drug monitoring; traceability
    DOI:  https://doi.org/10.1002/jms.5168
  2. Anal Sci. 2025 Aug 18.
      Fatty acids (FAs) are essential molecules in biological systems and have crucial roles for fundamental components of cellular membranes, energy stores and mediators for cellular functions. The growing importance of FAs has also paid attention to analytical methods for the determination of FA contents in various samples accurately. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is a powerful tool due to less time for sample preparation and analysis. This analytical approach enables to quantify levels of FA and other lipid classes in various samples simultaneously without derivatization. In the present study, the effects of ammonium salts on the sensitivity of FAs in LC-MS/MS analysis were evaluated. First, authors performed the comparison of FA analysis with different mobile phases, which contain ammonium formate, ammonium fluoride, ammonium acetate or ammonium carbonate. Interestingly, the mobile phase containing ammonium fluoride enhanced the sensitivity of targeted FA species more than other ammonium salts. Second, the difference of quantativity of FA species was examined. It was indicated that both carbon chain length and the number of double bonds affect ionization of FA species. Finally, the method of FA analysis based on ammonium fluoride was applied to FA profiling of lotus root. It is indicated that mobile phase containing ammonium fluoride enabled to evaluate FA contents with highly sensitivity in LC-MS/MS analysis.
    Keywords:  Ammonium fluoride; Fatty acid; Liquid chromatography tandem mass spectrometry
    DOI:  https://doi.org/10.1007/s44211-025-00832-7
  3. Food Chem. 2025 Aug 05. pii: S0308-8146(25)03057-2. [Epub ahead of print]493(Pt 3): 145806
      A sensitive technique for quantitative analysis of four Alternaria toxins (tenuazonic acid (TeA), alternariol (AOH), tentoxin (TEN), and alternariol monomethyl ether (AME)) in cereals and cereal-based foods was developed, which combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online mixed-mode strong anion exchange (MAX) solid-phase extraction (SPE) cleanup. Online SPE cleanup came after sample preparation, which included extraction using 50 mM NaH2PO4 aqueous-MeOH-ACN (45:10:45, v/v) and high-speed centrifugation. For quantification, isotope-labeled internal standard calibration curves were created with linear regression coefficients greater than 0.9988. The matrix effect values of Alternaria toxins ranged from -18.2 % to +22.5 %. The limits of detection (LODs) of TeA, AOH, TEN, and AME for this approach were determined to be 0.3, 0.15, 0.06, and 0.02 μg/kg, respectively. The sensitivity of this method met the EU indicative levels for infant foods. The recoveries of Alternaria toxins in rice, wheat, and corn were ranged from 86.9 % to 112.0 %, 84.2 % to 102.8 %, and 85.8 % to 105.6 %, respectively. The inter- and intra-day RSDs were lower than 10.2 %. This analytical approach was successfully applied to determine Alternaria toxins in 24 cereal-based samples from Zhoushan, China.
    Keywords:  Alternaria toxins; Cereals and cereal-based foods; LC-MS/MS; Online SPE cleanup
    DOI:  https://doi.org/10.1016/j.foodchem.2025.145806
  4. J Chromatogr A. 2025 Aug 08. pii: S0021-9673(25)00623-5. [Epub ahead of print]1760 466278
      Chiral impurity analysis presents significant challenges due to limitations of detection methods. Levodopa (L-Dopa) is the primary medication for Parkinson's disease treatment for over 50 years. However, the detection of its chiral impurity is difficult due to the similarity of polarity. This study presents a novel analytical method for the quantitative detection of trace amount of d-Dopa, as well as other three impurities. The method demonstrated excellent chromatographic resolution for d-Dopa and l-Dopa (resolution = 4.15) and achieved a derivatization efficiency of 97 % using a chlorine-labeled chiral probe (D-BPCl) for chemical derivatization, followed by analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a conventional C18 column. Meanwhile, d-BPCl showed strong chiral selectivity on d-Dopa, and the ratio of mass spectrometric response for labeled d-Dopa to that of l-enantiomers was 2.78-fold under the same condition. The developed method demonstrates high sensitivity with detection limits of 7.88 μg l-1 for d-Dopa, and 9.85 μg l-1 for l-Dopa, with good linearity for every analyte (R2 > 0.99), recoveries ranging from 94.1 % to 106.8 %, and high repeatability with relative standard deviation (RSD) values predominantly below 5 %. Then the method was applied to analyze fifteen batches of levodopa tablets from five manufacturers. d-Dopa was successfully detected with the corresponding content ranging from 0.15 % to 0.44 %, which suggests that d-Dopa are more likely generated during the production process. The study underscores the significant safety concerns regarding Dopa-containing medications.
    Keywords:  D-Dopa; Derivatization; Levodopa tablets; Quantitative detection
    DOI:  https://doi.org/10.1016/j.chroma.2025.466278
  5. Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00820-7. [Epub ahead of print]1371 344426
       BACKGROUND: Lipidomics can provide critical insight into metabolic changes in health and disease, but faces challenges in sensitivity, lipid coverage, and annotation accuracy. To address these limitations, we optimized a liquid chromatography-mass spectrometry (LC-MS) method combining scheduled data-dependent acquisition (SDDA) and C30 column-based separations, aimed at improving global lipidomics for clinical diagnostics.
    RESULTS: Compared to conventional DDA and Intelligent Data Acquisition (AcquireX), SDDA demonstrated a 2-fold increase in number of lipids annotated, with a 2-fold higher annotation confidence (Grade A and B) of those lipids compared to DDA. The repeatability and analytical robustness of the method were thoroughly evaluated across different clinical blood matrices, i.e. serum, EDTA-plasma, and dried blood spots (DBS). Serum provided the highest repeatability and lipid coverage, with more than 2000 lipid species annotated. A proof-of-concept study assessing postprandial lipidomic changes in response to intake of a long-chain triglyceride fat emulsion was used to demonstrate the method's applicability in clinical lipidomics. The method detected significant changes in the levels of various lipids, including triacylglycerols, diacylglycerols, bile acids, phosphatidylethanolamines, and lyso-phosphatidylethanolamines, following lipid ingestion.
    SIGNIFICANCE AND NOVELTY: The optimized lipidomics method (C30-SDDA) enhances lipid coverage and annotation confidence, proving valuable for studying metabolic alterations and biomarker discovery using blood matrices. These findings underscore the clinical potential of this method for advancing diagnostics and personalized medicine.
    Keywords:  C30 chromatography; Clinical lipidomics; Global lipidomics; LC-MS; Scheduled DDA
    DOI:  https://doi.org/10.1016/j.aca.2025.344426
  6. Metabolomics. 2025 Aug 19. 21(5): 121
       BACKGROUND: Metabolomics is rapidly evolving, addressing analytical chemistry challenges in the qualification and quantitation of metabolites in extremely complex samples. Targeted metabolomics involves the extraction and analysis of target compounds, often present at extremely low concentrations, whilst untargeted metabolomics requires the use of sophisticated analytical techniques to deal with the simultaneous identification or quantitation of hundreds of compounds. Given the high energy consumption and excessive amounts of waste generated by metabolomics studies, greenness metrics are essential to account for sustainable development.
    AIM OF REVIEW: To determine the applicability of the Analytical GREEnness calculator (AGREE) in evaluating the analytical greenness of metabolomics methods. Specifically, the analytical protocols of 16 state-of-art metabolomics studies, including nine targeted and seven untargeted metabolomics studies, are fully dissected, and detailed greenness parameters for each procedure are rationally estimated.
    KEY SCIENTIFIC CONCEPTS OF REVIEW: The calculated AGREE metrics unequivocally show the main weaknesses of greenness in current research, and guidelines for sustainable practices in metabolomics are provided. The results indicate that offline sample preparation and the lack of automation and miniaturization are key areas that must be addressed to make metabolomics more sustainable. Important aspects that should be considered include the complexity of sample preparation procedures, the use of toxic reagents and derivatizing agents, the amount of waste generated, and sample throughput.
    Keywords:  Green analytical chemistry; Greenness metrics; Mass spectrometry; Metabolomics; NMR
    DOI:  https://doi.org/10.1007/s11306-025-02323-2
  7. J Am Soc Mass Spectrom. 2025 Aug 19.
      In this paper, we present the Multipass CCS Refiner, an R application for calibrating multipass collision cross section (CCS) measurements with cyclic ion mobility spectrometry (cIMS). The Multipass CCS Refiner combines the necessary tools for calculating accurate CCS measurements from multipass cIMS experiments into a user-friendly web-based Shiny interface accessible regardless of programming knowledge. The application provides a suite of functions for calculating accurate arrival times, automated pass counting approximation, correction of arrival time perturbations during separation, constructing calibration curves unique to specific instrument settings, separating fine mobility features, and a variety of visualization tools. Code for the application is structured to be easily modified to meet the user's needs. The Multipass CCS Refiner implements the calibration approach described by Lin and Costello to reduce the need for carefully selected separation times while accounting for artifacts underlying fluctuations in measured ion arrival times. The application is showcased with downloadable data files of commonly used standards, which can be run in the app directly or edited with user experimental data.
    Keywords:  collision cross section calibration; cyclic ion mobility spectrometry; multipass CCS Refiner; traveling-wave perturbation-correction
    DOI:  https://doi.org/10.1021/jasms.5c00199
  8. Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00822-0. [Epub ahead of print]1371 344428
       BACKGROUND: Perturbed lipid metabolism in cellular environment plays an essential role in the progression of peripheral neuropathy, such as Charcot-Marie-Tooth disease type 1A (CMT1A). Current deep-profiling of lipidome still has certain limitations for high-throughput analysis of clinical samples, such as low sensitivity, extensive requirement of instrument modification, technician-dependent pretreatment and lack of method-adapted data-processing software.
    RESULTS: Herein, a new generation aza-Prilezhaev aziridination (APA) reagent has been designed for coupling with high-resolution liquid chromatography mass spectrometry (LC-MS) to conduct comprehensive untargeted deep lipidomics of CMT1A disease. It facilitates large-scale lipid structural annotations over a wide polarity range in a single run with high-sensitivity. With the developed method, an extensive library containing information on retention time, m/z, and MS/MS down to CC location levels for 393 unsaturated lipids of CMT1A was created. Significant disease-related differences in CC isomer profiles were observed from the comparison of plasma between CMT1A patients (N = 9) and controls (N = 8). Specifically, 40 different lipids were identified, predominantly comprising n-6, n-7, and n-9 CC positional isomers.
    SIGNIFICANCE: This innovative methodology would offer substantial potential to enhance diagnosis and therapeutic strategies for CMT1A and other diseases based on lipid isomerization information.
    Keywords:  Automatic data-processing; Aziridination; CC location; Charcot-Marie-Tooth disease; Lipidomics; Liquid chromatography mass spectrometry
    DOI:  https://doi.org/10.1016/j.aca.2025.344428
  9. ChemSusChem. 2025 Aug 18. e2501158
      A workflow for the quantification of electrolyte residues, including linear and cyclic carbonates, conducting salt and selected degradation products from shredded lithium ion battery material black mass, is developed. Therefore, a liquid chromatography method hyphenated to a tandem mass spectrometer is set up which is capable of separating and reliably quantifying standard organic electrolyte compounds showing low limits of quantification and detection. For the quantification of ionic species, ion chromatography with a conductivity detector is used. The combination of data sets shows that up to 5.5 wt% of organic compounds and 2.46 wt% of ionic species are extracted from the black mass with ethylene carbonate and hexafluorophosphate (PF6 -) being the most prominent species. The use of protic solvents, such as water and methanol, results in degradation reactions forming ethylene glycol and fluoride (F-) respectively. Three different extraction methods are evaluated for their applicability in a quantitative analysis setup. The results demonstrate that shake extraction using the aprotic acetonitrile is the most suitable sample preparation technique with over 90% extraction after a single cycle without introducing solvolysis nor thermal degradation.
    Keywords:  analytical methods; batteries; black mass; electrolyte residues; extraction methods
    DOI:  https://doi.org/10.1002/cssc.202501158
  10. Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00817-7. [Epub ahead of print]1371 344423
       BACKGROUND: We have developed a new class of dual polarity molecules for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) capable of acquiring 5 μm pixel sizes with high sensitivity toward polar lipids and metabolites. Aminated cinnamic acid analogs (ACAAs) are vacuum stable, have high extinction coefficients at 355 nm, are highly sensitive to polar lipids, have low toxicity, and are affordable. Current molecules used for high spatial resolution MALDI IMS of polar lipids have shown great success, but are plagued with issues such as low sensitivity at high spatial resolution, vacuum instability, and/or high toxicity.
    RESULTS: ACAAs were evaluated as MALDI matrices, testing them for vacuum stability, absorption at 355 nm, crystal size, sensitivity, and molecular coverage. Among them, 4-aminocinnamic acid (ACA) and 4-(dimethylamino)cinnamic acid (DMACA) were found to perform better than conventional MALDI matrices for lipid IMS experiments. ACA generated fewer in-source fragments due to its high extinction coefficient at 355 nm. This leads to better discernment of thermally labile molecules such as gangliosides compared to typical 'soft' ionization matrices like DHA using murine brain tissue. On the other hand, DMACA showed better optical properties than ACA, giving it higher sensitivity from many lipid classes, such as phospholipids and sulfatides. DMACA outperformed DAN and DHA at their individually optimized laser power at small pixel sizes (≤10 μm). DMACA also allows for lower laser power to be used without compromising sensitivity, which reduced the laser spot size at the sample surface from ∼6 μm to ∼4.5 μm without hardware modifications.
    SIGNIFICANCE: Improved sensitivity and absorption efficiency at 355 nm allow for 5 μm pixel size MALDI IMS without oversampling while maintaining high S/N on commercial mass spectrometry platforms. Performing MALDI experiments at reduced laser energies minimizes tissue damage, enabling advanced multimodal MALDI IMS studies to be performed on single tissue sections. Comparisons and optimized MALDI IMS methods were performed on murine tissues and human kidney samples as part of the Human Biomolecular Atlas Program.
    Keywords:  4-Aminocinnamic acid (ACA); 4-Dimethylaminocinnamic acid (DMACA); Chemical matrix; High spatial resolution; Imaging mass spectrometry; Lipids; Matrix-assisted laser desorption/ionization (MALDI); timsTOF
    DOI:  https://doi.org/10.1016/j.aca.2025.344423
  11. Prep Biochem Biotechnol. 2025 Aug 19. 1-6
      Affinity chromatography-based methods for immunoglobulin G quantification present an attractive alternative to widely used nephelometry due to their simplicity, speed, and compatibility with various sample types. This study validates the efficient analytical use of commercially available POROS CaptureSelect FcXP affinity resin, a stationary phase optimized for fast recombinant human IgG purification. The analytical method was validated in a simple bind-elute mode with a cycle time of 5 minutes. Linearity was confirmed in a range of 4 µg (lower limit of quantification) to 260 µg, allowing direct quantification of IgG from human plasma without further dilution. The method demonstrated excellent precision, with %RSD values ranging from 5% to 1% and satisfactory recovery 99% on average, respectively. By decreasing the residence time from 0.2 minutes to 0.1 minutes, a cycle time of 2.5 minutes was achieved, demonstrating a powerful chromatographic method for fast and reliable IgG quantification. This method offers significant advantages over nephelometry, including reduced analysis time and enhanced compatibility with high-throughput workflows.
    Keywords:  Antibody; Cohn fractionation; Ethanol fractionation; IVIG; VHH
    DOI:  https://doi.org/10.1080/10826068.2025.2547195
  12. Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00819-0. [Epub ahead of print]1371 344425
       BACKGROUND: Mass spectrometry imaging (MSI) and Raman spectroscopy imaging are two chemical imaging techniques that can reveal and visualize the chemical distributions in thin tissue sections. The two techniques are complementary with Raman spectroscopy detecting the Raman shift of molecular bonds while MSI detects mass-to-charge of ionized molecules. Furthermore, MSI requires molecules to be desorbed, ablated or sputtered from the surface, making Raman spectroscopy less destructive in comparison. With a multimodal analysis workflow, the advantages of both techniques can be combined to yield enhanced chemical information of surfaces, such as thin tissue sections. Here, we combine the two imaging modalities and map the chemical signature in the exact same location on thin tissue section to uncover MSI derived chemical alterations in tissue.
    RESULTS: In this study, we present a multimodal workflow integrating pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) MSI and Raman spectroscopy on the same tissue section. This requires some adaptation of MSI to the sample restrictions of Raman. We demonstrate successful integration and the power of combined analysis in characterization of tissue chemistry. Raman spectroscopy proved highly effective for identifying solvent-induced chemical alterations in tissue caused by PA nano-DESI MSI with methanol or acetonitrile-based solvents. First, we describe chemical alterations connected to different sampling modes, such as different probe speeds and oversampling. Following, we characterize chemical changes in the resulting tissue exposed to up to five repeated imaging experiments. The results from the two modalities were in excellent agreement and showed that methanol desorbs more material from the tissue compared to acetonitrile, and that both solvents cause protein denaturation in the tissue.
    SIGNIFICANCE: This study presents the establishment of a multimodal workflow combining imaging with PA nano-DESI MSI and Raman spectroscopy from the exact same tissue sections and locations. The combination enabled chemical characterization of the tissue after MSI and revealed previously unknown alterations in tissue chemistry upon MSI. Overall, our findings offer new insights into how exposure to solvents impact tissue chemistry and highlight the potential of combining MSI with Raman spectroscopy for in depth chemical characterization of tissue sections.
    Keywords:  Extraction; Lipidomics; Metabolomics; Multimodal imaging; Nano-DESI; Raman; Tissue
    DOI:  https://doi.org/10.1016/j.aca.2025.344425
  13. Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00830-X. [Epub ahead of print]1371 344436
       BACKGROUND: Three-phase liquid membrane extraction (LME) of acids involves mass transfer from an acidified sample, through an organic liquid membrane into an alkaline aqueous acceptor. However, this approach presents challenges for acids with pKa > 9-10, as their efficient extraction often requires extreme pH conditions in the acceptor, which can compromise chemical stability and compatibility with chromatographic analysis. Alternatively, a polar organic solvent can be used as acceptor, but this may challenge the stability of the liquid membrane and the integrity of the extraction system.
    RESULTS: In this work, commercial 96-well plates were used for the extraction of nine weakly acidic model analytes (phenols and bisphenols). With an alkaline acceptor, the presence of a boundary layer between the liquid membrane and the acceptor hindered the extraction of the analytes, requiring the use of pH 13.0. To overcome this, an alternative system was developed with acceptor based on dimethyl sulfoxide diluted with pure water. In both systems, different liquid membranes were evaluated, and extraction kinetics were studied. Both systems were applied to human plasma and provided exhaustive extraction of the analytes. Under the final conditions, the DMSO-water acceptor system was evaluated obtaining satisfactory analytical parameters in terms of linearity (r2 > 0.990) and precision (RSD ≤15 %).
    SIGNIFICANCE: This new approach enhances the applicability of the three-phase aqueous-organic-organic system, making it milder towards the liquid membrane. It enables the extraction of the analytes as neutral species, broadening the scope of extractable compounds in the three-phase system. Additionally, it offers an alternative to the well-established ionization mode.
    Keywords:  Bisphenols; Liquid-phase microextraction; Phenols; Sample preparation; Supported liquid membrane
    DOI:  https://doi.org/10.1016/j.aca.2025.344436