bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–02–02
forty-one papers selected by
Sofia Costa, Matterworks
  1. Comprehensive untargeted polar metabolite analysis using solvent switching liquid chromatography tandem mass spectrometry.
  2. Extraction, Annotation, and Purification of Resin Glycosides from the Morning Glory Family (Convolvulaceae).
  3. Identification and Profiling of Primary Metabolites Through GC-MS and Associated Data Processing.
  4. Development of an accurate and sensitive assay for 2-methoxyestradiol using derivatization and liquid chromatography-tandem mass spectrometry.
  5. Determination of 115 Pesticide Residues in Textiles by Liquid Chromatography-Tandem Mass Spectrometry.
  6. Development and validation of a sensitive LC-MS/MS assay for determination of upadacitinib in human plasma and its application in patients with inflammatory bowel disease.
  7. Collection of optimizations for untargeted metabolomics analysis of Leishmania promastigotes using gas chromatography-mass spectrometry.
  8. Simultaneous determination of busulfan, fludarabine, phenytoin, and posaconazole in plasma from patients undergoing hematopoietic stem cell transplantation.
  9. Ultrasensitive LC-MS/MS quantitation of the N-nitroso-dabigatran etexilate impurity in dabigatran etexilate mesylate using an electrospray ionization technique.
  10. Identification of Flavonoids Using UV-Vis and MS Spectra.
  11. Conjugation of Short-Chain Fatty Acids to Bicyclic-Amines for Analysis by Liquid Chromatography Tandem Mass Spectroscopy.
  12. Method Development and Clinical Utility for Simultaneous Measurement of 21-Deoxycortisol, 17-Hydroxyprogesterone, Cortisol, and Cortisone Levels in Human Plasma Using UHPLC-MS/MS.
  13. Expanding the chemical coverage of polar compounds in water analysis by coupling supercritical fluid with hydrophilic interaction chromatography high-resolution mass spectrometry.
  14. Staining Tissues with Basic Blue 7: A New Dual-Polarity Matrix for MALDI Mass Spectrometry Imaging.
  15. Protocol for Oligonucleotides Characterization Using Hydrophilic Interaction Chromatography.
  16. A High-Throughput Integrated Nontargeted Metabolomics and Lipidomics Workflow Using Microelution Enhanced Matrix Removal-Lipid for Comparative Analysis of Human Maternal and Umbilical Cord Blood Metabolomes.
  17. Systematic Profile of Oxylipins in Myocardial Infarction by Liquid Chromatography-Tandem Mass Spectrometry.
  18. High-Resolution Isolation of Natural Products: Efficient Combination of Dryload Injection and HPLC Gradient Transfer.
  19. Simultaneous quantification of siRNA antisense and sense strands by hybrid liquid chromatography-mass spectrometry.
  20. Enhanced bile acid detection and analysis in liver fibrosis with pseudo-targeted metabolomics.
  21. MetAssimulo 2.0: a web app for simulating realistic 1D & 2D Metabolomic 1H NMR spectra.
  22. Method development and validation for the simultaneous determination of 21 antiviral drugs by ultra-high performance liquid chromatography-tandem mass spectrometry in chicken muscle and liver.
  23. Direct Determination of Flavanone Isomers in Citrus Juice by Paper Spray Tandem Mass Spectrometry.
  24. Multidimensional library for the improved identification of per- and polyfluoroalkyl substances (PFAS).
  25. Sample Preparation of Caenorhabditis elegans for GC-MS-Based Metabolomics in Toxicity Assessment.
  26. Identification of Primary Metabolites in 1H NMR-Based Metabolomics of Plants.
  27. Development of a novel HPLC-HRMS method for quantitative analysis of resorcinol in urine: Application to hairdressers' occupational exposure.
  28. A Fast-Pass, Desorption Electrospray Ionization Mass Spectrometry Strategy for Untargeted Metabolic Phenotyping.
  29. Development and application of an efficient, accurate, and environmentally friendly liquid chromatography-tandem mass spectrometry method for the determination of five Alternaria toxins in wheat.
  30. msiFlow: automated workflows for reproducible and scalable multimodal mass spectrometry imaging and microscopy data analysis.
  31. Using DIMet for Differential Analysis of Labeled Metabolomics Data: A Step-by-step Guide Showcasing the Glioblastoma Metabolism.
  32. Fentanyl and Fentanyl Analog Screening Using ASAP-MS With LiveID Confirmation.
  33. Optimization and Validation of an Ultra-Performance Liquid Chromatography with Quadrupole Detector Mass Spectrometry Quantification Method for the Simultaneous Detection of Tazarotene and Tazarotenic Acid in Porcine Skin: An In Vitro Study.
  34. Analysis and Identification of Polyphenols in Fragaria, Rubus, and Ribes Species by Nontargeted UHPLC-PDA-MS Metabolite Profiling.
  35. Stereoselective Reaction Enabling Simultaneous Analysis of Carbon-Carbon Double-Bond Configuration and the Position of Monounsaturated Fatty Acids through UHPLC-ESI-MRM-MS.
  36. Exploring the plant lipidome: techniques, challenges, and prospects.
  37. MetaboLINK is a novel algorithm for unveiling cell-specific metabolic pathways in longitudinal datasets.
  38. Unveiling Impurity Profiling of Synthetic Pathways of Organophosphorus Chlorpyrifos Through LC-HRMS Metabolomics-Based Approaches.
  39. Two-Dimensional Liquid Chromatography-Isotope Ratio Mass Spectrometry Opens New Possibilities in the Field of Compound-Specific Stable Isotope Analysis.
  40. Analytical method development and validation for monosaccharide profiling in Lignosus rhinocerotis using rP-HPLC.
  41. QuanFormer: A Transformer-Based Precise Peak Detection and Quantification Tool in LC-MS-Based Metabolomics.
  1. Talanta. 2025 Jan 16. pii: S0039-9140(25)00096-7. [Epub ahead of print]287 127610
    Comprehensive untargeted polar metabolite analysis using solvent switching liquid chromatography tandem mass spectrometry.
    Jake P Violi, Connor R Philips, David S Gertner, Mika T Westerhausen, Matthew P Padula, David P Bishop, Kenneth J Rodgers.
      Metabolomics analyses enable the examination and identification of endogenous biochemical reaction products, revealing information on the metabolic pathways and processes active within a living cell or organism. Determination of metabolic shifts can provide important information on a treatment or disease. Unlike other omics fields that typically have analytes of the same chemical class with common building blocks, those that fall under the nomenclature of metabolites encompass a wide array of different compounds with very diverse physiochemical properties. Development of a comprehensive metabolomic pipeline therefore can be a troublesome and complicated process for the analyst. Often single liquid chromatography-mass spectrometry methods on unfractionated samples are carried out in order to be time-efficient, however this could potentially produce data with a low number of identifiable metabolites. In the present studies, we developed a comprehensive polar metabolomics pipeline for cell-based metabolomics. SH-SY5Y neuroblastoma cells were selected as the sample matrix for method development since they are one of the most widely used cell lines for human neurotoxicity studies. This was accomplished by investigating and optimising different mass spectrometry source and chromatographic conditions to enhance the signal of polar metabolites. Optimised hydrophilic interaction liquid chromatography (HILIC) based metabolomic methods at different pH values were examined in positive, negative, and polarity switching modes to determine which combination yielded the highest number of confidently identified metabolites. Additionally, the use of sequentially running two methods was also compared to determine the degree of overlap and whether there is merit in running two separate methods on one sample. It was determined that solvent switching between two optimised methods, acidic chromatographic conditions in positive mode and basic chromatographic conditions in negative mode, yielded the highest number of unique identifiable metabolites. This could be run in a single analytical batch due to the large pH range of the column. A quick switch method in-between each method allowed both conditioning the column and preparation of the MS source conditions for the sequential method.
    Keywords:  Chromatography; HILIC; LC-MS; Metabolomics; Method development; Optimisation
    DOI:  https://doi.org/10.1016/j.talanta.2025.127610
  2. Methods Mol Biol. 2025 ;2895 177-200
    Extraction, Annotation, and Purification of Resin Glycosides from the Morning Glory Family (Convolvulaceae).
    Lars H Kruse, Alexandra A Bennett, Vishwa J Baruah, Mohammad Irfan, Gaurav D Moghe.
      The metabolic diversity of plants, comprising over a million different metabolites across the plant kingdom, harbors enormous potential for pharmaceutical and biotechnological applications. Resin glycoside (RG) acylsugars from the Convolvulaceae are of interest due to their medicinal and agricultural potential. However, understanding the biological relevance of RGs is challenging as they exhibit a high lineage-specific structural diversity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with computational peak annotation can provide insights into this diversity. Here, we present a comprehensive protocol for the characterization of RG diversity using a sensitive LC-MS/MS instrument, a knowledge-based computational pipeline, and a web tool for peak annotation. The described experimental approach provides a step-by-step guide for RG sampling, extraction, purification for downstream analyses such as bioassays, and structural annotation using LC-MS/MS and computational metabolomics. The protocol focuses on qualitative analysis for putative annotation (Annotation Level 2 as defined by the Metabolomics Standards Initiative) of RGs and can serve as a valuable template for researchers exploring plant metabolic diversity beyond RGs and acylsugars.
    Keywords:  Acylsugars; Annotation Level 2 prediction; Computational metabolomics; Convolvulaceae; LC-MS; Morning glories; Plant metabolic diversity; Resin glycosides; Tandem mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-4350-1_13
  3. Methods Mol Biol. 2025 ;2895 99-109
    Identification and Profiling of Primary Metabolites Through GC-MS and Associated Data Processing.
    Minki Shim, Yooseong Jeong, Dong-Kyu Lee.
      This chapter presents a comprehensive approach to profiling plant-derived primary metabolites using metabolomics, highlighting its critical role in decoding the biosynthesis of bioactive plant compounds. It details the utilization of gas chromatography-mass spectrometry (GC-MS) for the effective analysis and profiling of these metabolites. The process, encompassing extraction methods, chemical derivatization, and data processing, is thoroughly outlined. This methodology outlines comprehensive procedures for each stage of the workflow, encompassing metabolite extraction, GC-MS analysis, and data alignment, to produce a metabolomics dataset.
    Keywords:  Data processing; Gas chromatography-mass spectrometry; Metabolite identification; Metabolomics; Plant primary metabolites
    DOI:  https://doi.org/10.1007/978-1-0716-4350-1_8
  4. Pract Lab Med. 2025 Apr;44 e00447
    Development of an accurate and sensitive assay for 2-methoxyestradiol using derivatization and liquid chromatography-tandem mass spectrometry.
    Koji Takahashi, Masaki Takiwaki, Seketsu Fukuzawa, Yoshikuni Kikutani, Kentaro Abe, Tatsuya Higashi, Hironori Kobayashi.
      2-Methoxyestradiol (2ME) is involved in the pathogenesis of preeclampsia and antitumor activity. In addition to its low concentration in healthy human serum, presence of isomers makes quantification of 2ME for clinical research and laboratory medicine difficult. The objective of this study was to develop a highly sensitive and accurate method for quantifying 2ME using LC-MS/MS combined with derivatization with 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F). This approach significantly enhanced the detectability of 2ME in positive electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and enabled the chromatographic separation of 2ME from isomeric metabolites possessing a methoxy group, including 4-methoxyestradiol, 3-O-methyl 2-hydroxyestradiol, and 3-O-methyl 4-hydroxyestradiol (3M4OH). Although the derivatized 2ME and 3M4OH were closely eluted under the optimized LC conditions, the different fragmentation patterns of these isomers during MS/MS allowed their distinction. The lower limit of quantification for 2ME was 2.5 pg/mL, indicating a satisfactory sensitivity. These findings demonstrated that this LC-MS/MS method combined with the MPDNP-F derivatization can provide accurate and highly sensitive quantification of 2ME.
    Keywords:  2-Methoxyestradiol; Estrogen; LC-MS/MS; MPDNP-F; derivatization
    DOI:  https://doi.org/10.1016/j.plabm.2024.e00447
  5. Rapid Commun Mass Spectrom. 2025 May 15. 39(9): e9992
    Determination of 115 Pesticide Residues in Textiles by Liquid Chromatography-Tandem Mass Spectrometry.
    Ying Li, Xiwen Ye, Xin Luo, Wenxuan Zhu, Zhixu Tang, Zengyuan Niu.
      The presence of pesticide residues in textiles poses a risk to human health. We established a robust and high-throughput liquid chromatography-tandem mass spectrometry method for the determination of 115 pesticide residues in textiles. In this study, we evaluated high-performance liquid chromatography-tandem mass spectrometry conditions and sample extraction methods, including separation performance of different columns, mass conditions, extraction solvent, and extraction time. Finally, we established the method as follows: After ultrasonic extraction with methanol, we blew the sample with nitrogen to dry and then took it to a fixed volume by a specific solvent. We used a C18 reversed-phase chromatographic column and detected the samples in the multiple reaction monitoring mass mode. And we verify the limit of detection (LOD), limit of quantitation (LOQ), linearity, recovery, and precision. The LOD and LOQ of the method was 10 and 20 μg/kg separately; the recoveries ranged from 71.3% to 118.4%; and the relative standard deviation was 0.2%-19.9%. We verified the applicability of the developed protocol through the analysis of 21 real textile products.
    Keywords:  liquid chromatography; pesticide residues; tandem mass spectrometry; textiles
    DOI:  https://doi.org/10.1002/rcm.9992
  6. J Pharmacol Toxicol Methods. 2025 Jan 23. pii: S1056-8719(25)00001-2. [Epub ahead of print]131 107581
    Development and validation of a sensitive LC-MS/MS assay for determination of upadacitinib in human plasma and its application in patients with inflammatory bowel disease.
    Jing-Jing Wu, Lang Lin, Jia-Jia Yan, Jia-He Kong, Qiao-Lan Xuan, Xiang Gao, Kang Chao, Xia Zhu.
       BACKGROUND: Upadacitinib is a selective Janus kinase (JAK) 1 inhibitor approved by the Food and Drug Administration for the treatment of moderate-to-severe inflammatory bowel disease (IBD). We aimed to establish and validate a method for determining Upadacitinib in patients with IBD by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
    METHODS: The mobile phase was 0.1 % formic acid: acetonitrile (35:65, v/v) at a flow rate of 0.40 mL/min. Upadacitinib and its internal standard Upadacitinib 15N, d2 were separated by a Waters Xbridge BEH C18 column (4.6 × 100 mm, 2.5 μm) and subjected to mass analysis using positive electrospray ionization (ESI).
    RESULTS: The calibration range of Upadacitinib was 0.5-200 ng/mL with the correlation coefficient r2 ≥ 0.99. Accuracies ranged from -9.48 % ∼ 8.27 % and the inter- and intra-day precisions were less than 15 % for all analytes in quality control samples. There was no significant matrix effect. The range of extraction recoveries was 87.53-93.47 % for all analytes. Twenty-one plasma samples were obtained from the sixth affiliated hospital of Sun Yat-sen University. The median plasma concentration of Upadacitinib was 7.32 (0.56-26.78) ng/mL.
    CONCLUSION: This newly developed method is sensitive, simple, and successfully applied in determining Upadacitinib in IBD patients to provide reference for safe and effective drug administration in clinical practice.
    Keywords:  Inflammatory bowel disease; LC–MS/MS; Therapeutic drug monitoring; Upadacitinib
    DOI:  https://doi.org/10.1016/j.vascn.2025.107581
  7. Talanta. 2025 Jan 20. pii: S0039-9140(25)00089-X. [Epub ahead of print]287 127603
    Collection of optimizations for untargeted metabolomics analysis of Leishmania promastigotes using gas chromatography-mass spectrometry.
    Gisele André Baptista Canuto, Fabiane Dörr, Ernani Pinto, Maria Júlia Manso Alves, João Pedro Simon Farah, Marina Franco Maggi Tavares.
      There is no consensus in the literature regarding the ideal protocol for obtaining and preparing cell samples for untargeted metabolomics. Nevertheless, the procedures must be carefully evaluated for proper and reliable results for each organism under study. This work proposes a novel protocol for determining intracellular metabolites in Leishmania promastigotes and is fully optimized for application in conjunction with gas chromatography-mass spectrometry platforms. Sample harvesting consisted of stopping metabolic activity by placing the parasite cells in a dry ice bath and removing extracellular interferants with two wash steps using cold PBS. The extraction is carried out with 1.0x108 promastigotes per sample using a mixture of cold 1:1 methanol:water and ultrasound mixing (1 min at 30 % power). Dried extracts were derivatized by oximation (at room temperature for 90 min), followed by silylation (at 40 °C for 30 min). The method developed here can cover a wide range of the Leishmania parasite metabolome, including amino acids and derivatives, organic and fatty acids, carbohydrates and derivatives, and steroids.
    Keywords:  Cellular metabolomics; GC-MS; Intracellular extraction; Leishmania infantum; Sample harvesting
    DOI:  https://doi.org/10.1016/j.talanta.2025.127603
  8. J Pharm Biomed Anal. 2025 Jan 22. pii: S0731-7085(25)00024-X. [Epub ahead of print]257 116683
    Simultaneous determination of busulfan, fludarabine, phenytoin, and posaconazole in plasma from patients undergoing hematopoietic stem cell transplantation.
    Xijuan Jiang, Jia Liu, Xuan Feng, Weijing Ding, Yu Han, Yabin Qin, Yile Zhao.
      A simple, fast, sample-saving, and sensitive liquid chromatography-tandem mass spectrometry method was established with a linear range adjusted by in-source collision-induced dissociation. Notably, this could simultaneously determine busulfan, fludarabine, phenytoin, and posaconazole in plasma from children, each having unique physical and chemical properties. The procedure necessitated only 20 μL of plasma and involved a simple protein precipitation process. Chromatographic separation was accomplished on a reversed-phase column (C18, 50 × 2.1 mm, 2.6 μm) through gradient elution utilizing water (containing 0.1 % formic acid and 2 mM ammonium acetate) and acetonitrile (containing 0.1 % formic acid) as the mobile phase. An injection volume of 2 μL was utilized, with a total run time of 3.6 min. Mass spectrum acquisition was performed on a Triple Quad™ 4500MD tandem mass spectrometer with an electrospray ionization source in positive mode. Moreover, in-source collision-induced dissociation was used to adjust the linear range of phenytoin due to its excessive response. The calibration curves ranged from 20 to 2560 ng/mL for busulfan, 10-1280 ng/mL for fludarabine, 0.4-51.2 μg/mL for phenytoin, and 0.1-12.8 μg/mL for posaconazole, with mean r2 greater than 0.997. In addition, the method underwent rigorous validation following the European Medicines Agency guidelines, demonstrating exceptional accuracy (90.5 %-106.7 %) and precision (2.0 %-13.0 %). Furthermore, its applicability to atypical matrices, including hemolytic and hyperlipidemic plasma, was thoroughly assessed. As such, this approach was effectively utilized for the therapeutic drug monitoring of busulfan, fludarabine, phenytoin, and posaconazole for children undergoing hematopoietic stem cell transplantation.
    Keywords:  Busulfan; Fludarabine; In-source collision-induced dissociation; LC-MS/MS; Phenytoin; Posaconazole
    DOI:  https://doi.org/10.1016/j.jpba.2025.116683
  9. Anal Methods. 2025 Jan 31.
    Ultrasensitive LC-MS/MS quantitation of the N-nitroso-dabigatran etexilate impurity in dabigatran etexilate mesylate using an electrospray ionization technique.
    Anant D Yadav, Ashish C Alaspure, Sandesh R Lodha, Amrutlal L Prajapat, Anil H Gore.
      Nitrosamine impurities, particularly nitrosamine drug substance-related impurities (NDSRIs), pose significant health risks due to their potential mutagenicity and carcinogenicity. Consequently, stringent regulatory guidelines have been established for their detection and quantification in pharmaceutical products. This study presents a simple, robust, and ultrasensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the N-nitroso-dabigatran etexilate (NDE) impurity in the drugs and capsules of dabigatran etexilate (DEM) using an electrospray ionization technique. Using a gradient elution program, with mobile phase A consisting of 5 mM ammonium formate buffer (pH 7.0 ± 0.1) and mobile phase B 100% acetonitrile, chromatographic separation was achieved on a Sapphirus C18 HP-classic column (250 mm × 4.6 mm × 5 μm). The system utilized an Agilent 1290 infinity series LC with a 6470B LC/TQ tandem mass spectrometer, operating at a 0.6 mL min-1 column flow rate. Detection and quantitation of the N-nitroso dabigatran etexilate (NDE) impurity were performed in positive ESI mode using multi-reaction monitoring (MRM). The validation of the method adhered to the ICH Q2 (R2) guidelines, showing excellent signal-to-noise ratios for the detection and quantification limits, with linearity observed (correlation coefficient > 0.99) over a range of 18-120 ppb. Accuracy was confirmed through recovery studies, yielding satisfactory results between 80 and 120% of spiked concentrations. This validated LC-MS/MS method is suitable for routine and stability analysis of N-nitroso-dabigatran etexilate (NDE) impurities in both drug substance and finished capsules.
    DOI:  https://doi.org/10.1039/d4ay01710b
  10. Methods Mol Biol. 2025 ;2895 111-135
    Identification of Flavonoids Using UV-Vis and MS Spectra.
    Özlem Erol, Sandra Irmisch.
      Flavonoids are a group of specialized metabolites that are ubiquitously found within the plant kingdom. While they fulfill various important functions within the plant, they are also utilized by humans in a variety of different fields such as medicine, food science, and agriculture. Thus, to elucidate the chemical composition of any given plant extract, extraction and identification of flavonoids are of high interest. In this chapter, we outline a general approach for analyzing flavonoids in complex plant extracts. Using blueberry as an example, we start by describing the extraction of plant material, followed by simple spectrophotometric measurements and thin layer chromatography (TLC), to get first insights into flavonoid content and composition. Furthermore, we describe a standard protocol for liquid chromatography-mass spectrometry (LC-MS) for the identification of specific flavonoids. By utilizing and combining the described techniques, general analysis of flavonoid content, composition, as well as identification of specific compounds can be realized within a complex metabolic mixture.
    Keywords:  Flavonoids; Mass Spectrometry (MS); Thin Layer Chromatography (TLC); UV spectroscopy; Ultra-High-Performance Liquid Chromatography (UHPLC)
    DOI:  https://doi.org/10.1007/978-1-0716-4350-1_9
  11. Molecules. 2025 Jan 16. pii: 341. [Epub ahead of print]30(2):
    Conjugation of Short-Chain Fatty Acids to Bicyclic-Amines for Analysis by Liquid Chromatography Tandem Mass Spectroscopy.
    Daniel N Darlington.
      Conjugation of short-chain fatty acids (SDFAs) to amines containing ring structures allows for better measurement by liquid chromatography tandem mass spectroscopy (LC-MS/MS). However, collision-induced dissociation (CID) results in breaking the conjugate back to the original SCFA and amine. We therefore set out to find an amine that would remain on the SCFA after CID and create a unique daughter for selectivity of measurement. Of twenty-seven amines with ring structures, we found four that contain bicycle-type structures (two rings connected by a carbon) with nitrogen in the second ring. CID removes the second ring at the nitrogen, leaving the first ring on the daughter. Of the four amines, 4-(pyrrolidine-1-ylmethyl) benzylamine (4PyBA) showed the strongest conjugation. Conjugation of 4PyBA to SCFA (C3-C6), their isomers and their phenylated versions (and isomers) resulted in good chromatographic peaks and separation. CID resulted in unique daughters that allowed for selectivity of measurement. Using this method, standard curves were generated that show good linearity (r2 > 0.99) in the nM and μM range with lower limits of detection between 40 and 229 nM for a 10 μL sample. Finally, we used this method to measure SCFA in plasma, liver, platelets, and red blood cells, demonstrating its use in biological systems. Because SCFAs are an index of microbiome diversity in the gastrointestinal track, this method will allow us to study changes in SCFAs and the microbiome in pathologic conditions including trauma, hemorrhage, and sepsis.
    Keywords:  chromatography; conjugation; liver; mass spectroscopy; plasma; platelets; red blood cells; short-chain fatty acids
    DOI:  https://doi.org/10.3390/molecules30020341
  12. Adv Pharmacol Pharm Sci. 2025 ;2025 3859670
    Method Development and Clinical Utility for Simultaneous Measurement of 21-Deoxycortisol, 17-Hydroxyprogesterone, Cortisol, and Cortisone Levels in Human Plasma Using UHPLC-MS/MS.
    Syed N Alvi, Saleh Al Dgither, Ali Al-Odaib.
      A simple and efficient validated assay for quantifying 21-deoxycortisol (21-DOC), 17-hydroxyprogesterone (17-OHP), cortisol, and cortisone in human plasma has been developed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Analysis of plasma samples were performed on Atlantis dC18 (3 μm) column using a mobile phase of 20.0 mM ammonium acetate and acetonitrile (50:50, v : v) that was delivered at isocratic flow rate 0.3 mL/minute. After addition of d4-cortisol as an internal standard (IS), plasma samples containing 21-DOC, 17-OHP, cortisol, and cortisone were extracted with mixture of dichloromethane and tert-butylmethyl ether 1:2 (v/v). Analytes were detected and quantified in the positive ion mode of electrospray ionization using multiple reaction monitoring transition set at mass to charge (m/z): 347.17 ⟶ 311.12, 331.17 ⟶ 96.97, 363.11 ⟶ 121.00, 361.18 ⟶ 163.11, and 367.19 ⟶ 121.24 for 21-DOC and 17-OHP, cortisol, cortisone, and cortisol-d4 (IS), respectively. The relationship between concentration and peak area response (analyte/IS) were linear over the range of 0.25-50, 0.5-100, 1-200, and 2-400 ng/mL for 21-DOC, 17-OHP, cortisone, and cortisol, respectively. The mean extraction recovery of the analytes was in the range of 83%-96%. The coefficient of variation within and between days was less than 13.6%, and the bias ranged from -9.2% to 12%. The measured level of cortisol, cortisone, and 17-OHP was in the range of 21.9-110, 4.33-12.71, and 0.37-1.4 ng/mL, respectively. Furthermore, the measured value of cortisone-cortisol ratio was in the range of 0.08-0.21.
    Keywords:  21-deoxycortisol and 17-hydroxyprogesterone; UHPLC-MS/MS; cortisol; cortisone; plasma
    DOI:  https://doi.org/10.1155/adpp/3859670
  13. Anal Chim Acta. 2025 Mar 01. pii: S0003-2670(25)00066-2. [Epub ahead of print]1341 343672
    Expanding the chemical coverage of polar compounds in water analysis by coupling supercritical fluid with hydrophilic interaction chromatography high-resolution mass spectrometry.
    A Cerrato, T Holmark, E Emke, E D Amato, A F G Gargano.
       BACKGROUND: Persistent and mobile organic compounds (PMOC) are of great concern for water quality and human health. The recent improvement and availability of high-resolution mass spectrometry in combination with liquid chromatography have widely expanded the potential of analytical workflows for their detection and quantitation in water. Given their high polarity, the detection of some PMOC requires alternative techniques to reversed-phase chromatography, such as hydrophilic interaction liquid chromatography (HILIC) and supercritical fluid chromatography (SFC). Unified chromatography (UC), an SFC gradient in which the state of the mobile phase changes continuously from supercritical to liquid at 100 % polar co-solvent, has shown potential for the analysis of compounds in a broad range of polarity, including very polar compounds.
    RESULTS: In the present study, for the first time, a UC-HILIC method coupled with high-resolution mass spectrometry was set up for PMOC analysis in water. SFC and HILIC gradients were run sequentially on the same bare-silica column, with the first separation running to 100 % modifier (UC) followed by a HILIC gradient transitioning to water. The UC and UC-HILIC gradients were previously optimized on a mix of 18 representative PMOC to assess solvent and mobile phase composition and for the instrumental system setup. The final method was employed for the analysis of water samples in comparison with a traditional reversed-phase separation, resulting in a significant increase in the number of annotated polar PMOC, including compounds listed in the Candidate List of substances of very high concern for Authorisation by the European Chemicals Agency.
    SIGNIFICANCE: The proposed approach represents a robust alternative to traditional methods for broadening the chemical space of separation and mass spectrometric detection. The introduction of the HILIC section of the gradient was necessary for the elution of strongly retained compounds on the silica phase, thus also reducing the amount of compounds that would be permanently bound onto the phase of the column, resulting in possible irreproducibility, pressure increase, and loss of efficiency in the compound separation.
    Keywords:  High-resolution mass spectrometry; Non-targeted screening; Persistent and mobile organic compounds; Supercritical fluid chromatography; Unified chromatography; Water analysis
    DOI:  https://doi.org/10.1016/j.aca.2025.343672
  14. Anal Chem. 2025 Jan 30.
    Staining Tissues with Basic Blue 7: A New Dual-Polarity Matrix for MALDI Mass Spectrometry Imaging.
    Michal Javorek, Michal Hendrych, Kateřina Ondráková, Jan Preisler, Antonín Bednařík.
      Obtaining high-quality matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) images and the reproducibility of the technique depend strongly on the sample preparation protocol. The most crucial part is the application of the MALDI matrix, which often relies on expensive spraying or sublimation coaters. In this work, we present a new dual-polarity matrix for MALDI mass spectrometry imaging (MSI): Basic Blue 7 (BB7), which belongs to the group of triarylmethane dyes. Thanks to its good solubility in water, this matrix allows a quick and simple sample preparation protocol without the need for sophisticated spraying or sublimation instrumentation: dipping the glass with tissue into the dye solution. This technique closely resembles the staining methods employed in classical histopathology. The technique is demonstrated on MSI of lipids in mouse brain sections in positive and negative ion modes using a subatmospheric pressure MALDI source coupled with an orbital trap mass spectrometer. The results are compared with traditional matrices, such as 2,5-dihydroxybenzoic acid (DHB) and 1,5-diaminonaphthalene (DAN). BB7 excels, especially in negative ion mode, offering low background signals and high signal intensities of many lipid classes. Furthermore, the stained tissue can simply be inspected visually and allows basic histopathology annotation prior to MSI. Here, we demonstrate that staining offers excellent image quality, reproducible sample preparation, and the potential for automation and utilization for high spatial resolution MSI.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05244
  15. J Sep Sci. 2025 Jan;48(1): e70088
    Protocol for Oligonucleotides Characterization Using Hydrophilic Interaction Chromatography.
    Davy Guillarme, Szabolcs Fekete, Sylwia Studzińska.
      Oligonucleotides (ONs) are an increasingly popular category of molecules in the pharmaceutical landscape, particularly attractive for the treatment of genetic and rare diseases. However, analyzing these molecules presents significant challenges, due to their highly hydrophilic nature, multiple negative charges, and the presence of closely related impurities resulting from the complex solid-phase synthesis process. Ion pairing reverse-phase liquid chromatography (IP-RPLC) is the preferred technique for ONs analysis but is not ideal for mass spectrometry (MS) coupling. Consequently, there is a growing interest in exploring alternative strategies with hydrophilic interaction chromatography (HILIC) emerging as one of the most promising options. As HILIC is not yet fully established for the analysis of ONs, we have prepared this protocol paper to facilitate entry into this field. It not only provides best practices, opportunities, and potential advantages but also caveats and other important considerations for using HILIC to characterize ONs. The paper addresses the selection of stationary and mobile phases, optimization of gradient conditions, MS coupling, and key aspects to consider when manipulating ON samples. We hope this protocol will help establish HILIC as a more universal solution for ONs analysis.
    Keywords:  adsorption; hydrophilic interaction chromatography; mass spectrometry; mobile phase; protocol; stationary phase
    DOI:  https://doi.org/10.1002/jssc.70088
  16. Anal Chem. 2025 Jan 30.
    A High-Throughput Integrated Nontargeted Metabolomics and Lipidomics Workflow Using Microelution Enhanced Matrix Removal-Lipid for Comparative Analysis of Human Maternal and Umbilical Cord Blood Metabolomes.
    Wenjie Wu, Ke Wang, Jianing Liu, Pui-Kin So, Ting-Fan Leung, Man-Sau Wong, Danyue Zhao.
      Sample pretreatment for mass spectrometry (MS)-based metabolomics and lipidomics is normally conducted independently with two sample aliquots and separate matrix cleanup procedures, making the two-step process sample-intensive and time-consuming. Herein, we introduce a high-throughput pretreatment workflow for integrated nontargeted metabolomics and lipidomics leveraging the enhanced matrix removal (EMR)-lipid microelution 96-well plates. The EMR-lipid technique was innovatively employed to effectively separate and isolate non-lipid small metabolites and lipids in sequence using significantly reduced sample amounts and organic solvents. Our proposed methodology enables parallel profiling of metabolome and lipidome within a single sample aliquot using ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Following method development and optimization with representative metabolites at levels comparable to those detected in human blood, the optimized workflow was applied to prepare metabolome-lipidome from maternal and umbilical cord-blood sera prior to comprehensive profiling using three different UHPLC columns. Results indicate that, compared with conventional two-step metabolomics-lipidomics sample pretreatment workflow, this new approach substantially reduces sample amount and processing time, while still preserving metabolite profiles and revealing additional MS features. Over 2500 metabolites were annotated in human sera with >1000 shared across maternal and cord blood. The shared metabolites are closely linked to various physiological functions, including nutrient transfer, hormonal regulation, waste product clearance, and metabolic programming, underscoring the significant impact of maternal metabolic activities on neonatal metabolic health. In summary, the proposed workflow enables efficient sample pretreatment for nontargeted metabolomics-lipidomics using one single sample while achieving broad metabolite coverage, highlighting its remarkable applicability in clinical and preclinical research.
    DOI:  https://doi.org/10.1021/acs.analchem.4c03222
  17. Rapid Commun Mass Spectrom. 2025 May 15. 39(9): e9999
    Systematic Profile of Oxylipins in Myocardial Infarction by Liquid Chromatography-Tandem Mass Spectrometry.
    Xin Tan, Hui Ni, Qin Li, Zhanglin Ma.
       RATIONALE: Oxylipins play an important role in inflammatory processes, accompanying the occurrence of myocardial infarction. Analyzing a wide panel of oxylipins derived from more polyunsaturated fatty acids may provide valuable information to elucidate the relationships between the signaling mediator profile and myocardial infarction comprehensively.
    METHODS: An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneously measuring 74 oxylipins in 50-μL human samples with a 10-min chromatographic run and easy operation has been developed and validated.
    RESULTS: Accuracy and precision were below 15% of the relative errors in 99% of quality control. Recoveries and matrix effect were considered acceptable. Potential effects of different collecting tubes were also assessed. We successfully utilized our approach to measure plasma samples obtained from 99 healthy individuals and 302 myocardial infarction patients.
    CONCLUSIONS: Profiles of oxylipins discovered potential biomarkers and clarified the pathological characteristics of oxylipin metabolism in myocardial infarction. Our approach was rapid, accurate, and precise, with high throughput, low sample volume, and easy operation, suitable for large-scale studies.
    Keywords:  UHPLC–MS/MS; healthy individuals; myocardial infarction; oxylipins
    DOI:  https://doi.org/10.1002/rcm.9999
  18. Methods Mol Biol. 2025 ;2895 31-45
    High-Resolution Isolation of Natural Products: Efficient Combination of Dryload Injection and HPLC Gradient Transfer.
    Emerson Ferreira Queiroz, Davy Guillarme, Jean-Luc Wolfender.
      The isolation of pure compounds from complex extracts is a crucial step in natural products (NPs) research. Historically, this process has been recognized to be slow and laborious. However, significant advancements have been made in isolation methods. Efficient separation conditions can now be efficiently determined at the analytical scale using high- or ultra-high-performance liquid chromatography and transferred to the preparative scale by chromatographic calculations. This ensures consistent selectivity at both scales, offering precise separation predictions. High-resolution chromatographic conditions at the preparative scale can be achieved through optimized sample preparation and dry load sample introduction. Monitoring chromatographic traces issued from ultraviolet (UV), mass spectrometry (MS), and/or universal detectors such as evaporative light-scattering detectors (ELSD) can precisely guide the isolation or trigger the collection of specific NPs with diverse structural scaffolds. These approaches can be applied across different scales depending on the amounts of NPs targeted for isolation. This chapter presents a detailed description of an isolation protocol, using the aforementioned methodologies.
    Keywords:  Dry load; Gradient transfer; High-resolution isolation; Natural products; Semi-preparative high-performance liquid chromatography; Ultra-high-performance liquid chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-4350-1_3
  19. Bioanalysis. 2025 Jan 31. 1-11
    Simultaneous quantification of siRNA antisense and sense strands by hybrid liquid chromatography-mass spectrometry.
    Karan Agrawal, Shaofei Ji, Wenying Jian.
       BACKGROUND: Most oligonucleotide bioanalytical assays currently only quantify the pharmacologically-active antisense strand, though there have been recent efforts to simultaneously quantify the sense strand using hybridization ELISA or solid phase extraction LC-MS. Hybrid LC-MS, which offers both high sensitivity and specificity unlike the currently used platforms, has not been applied to quantify both siRNA strands simultaneously.
    MATERIALS & METHODS: A hybrid LC-MS assay utilizing LNA capture probes was developed and applied to quantify both strands of a 21-mer lipid-conjugated siRNA (SIR-3) using tandem mass spectrometry (MS/MS). A similar approach using high-resolution mass spectrometry (HRMS) was also evaluated.
    RESULTS: The final LC-MS/MS method was capable of quantifying both strands of SIR-3 at concentrations between 0.600 and 1000 ng/mL in cynomolgus monkey tissue homogenates with acceptable accuracy and precision. The LC-HRMS assay demonstrated similar sensitivity and assay performance as the LC-MS/MS assay.
    CONCLUSIONS: Overall, this manuscript presents orthogonal methods to existing siRNA bioanalytical workflows that with high sensitivity and specificity can provide greater information about the concentration and biotransformation of an siRNA analyte.
    Keywords:  Hybridization; LC-MS; capture probe; locked nucleic acids; oligonucleotides; short interfering RNA (siRNA)
    DOI:  https://doi.org/10.1080/17576180.2025.2457894
  20. J Pharm Biomed Anal. 2025 Jan 21. pii: S0731-7085(25)00009-3. [Epub ahead of print]257 116668
    Enhanced bile acid detection and analysis in liver fibrosis with pseudo-targeted metabolomics.
    Zhizhi Hu, Jiaojiao Wei, Kua Dong, Linnan Li, Aizhen Xiong, Li Yang, Zhengtao Wang.
      Bile acids (BAs) are essential signaling molecules that engage in host and gut microbial metabolism, playing a crucial role in maintaining organismal stability. Liquid chromatography-mass spectrometry (LC-MS) is a widely employed technique for metabolite analysis in biological samples due to its high sensitivity, excellent specificity, and low detection limits. This method has emerged as the mainstream approach for the detection and analysis of BAs. Pseudo-targeted analysis combines the advantages of both untargeted and targeted metabolomics methodologies. In this study, we developed a comprehensive and rapid method for detecting and analyzing BAs using LC-MS technology, applied to liver samples from bile duct-ligated (BDL) mice exhibiting liver fibrosis. A self-constructed database containing 488 BAs was established, and raw data from universal metabolome standard (UMS) were acquired using UHPLC-Q/TOF-MS. A total of 172 BA compounds were characterized, including 74 free BAs and 158 BAs were successfully detected using the high-coverage assay established with UHPLC-QQQ-MS. This assay was employed in the BDL liver fibrosis mouse model, where statistical analysis tools identified 20 differential BAs in the livers of affected mice. The development of this rapid method signifies a substantial advancement in the field, illustrating its utility in identifying differential BAs and enhancing our understanding of liver fibrosis. Furthermore, the high-coverage assay's ability to accurately analyze a diverse range of BAs could substantially aid in diagnosing and treating liver diseases.
    Keywords:  Bile acids; Liver fibrosis model; Method development; Pseudo-targeted metabolomics; Quantitative
    DOI:  https://doi.org/10.1016/j.jpba.2025.116668
  21. Bioinformatics. 2025 Jan 25. pii: btaf045. [Epub ahead of print]
    MetAssimulo 2.0: a web app for simulating realistic 1D & 2D Metabolomic 1H NMR spectra.
    Yan Yan, Beatriz Jiménez, Michael T Judge, Toby Athersuch, Maria De Iorio, Timothy M D Ebbels.
      Metabolomics extensively utilizes Nuclear Magnetic Resonance (NMR) spectroscopy due to its excellent reproducibility and high throughput. Both one-dimensional (1D) and two-dimensional (2D) NMR spectra provide crucial information for metabolite annotation and quantification, yet present complex overlapping patterns which may require sophisticated machine learning algorithms to decipher. Unfortunately, the limited availability of labeled spectra can hamper application of machine learning, especially deep learning algorithms which require large amounts of labelled data. In this context, simulation of spectral data becomes a tractable solution for algorithm development.Here, we introduce MetAssimulo 2.0, a comprehensive upgrade of the MetAssimulo 1.0 metabolomic 1H NMR simulation tool, reimplemented as a Python-based web application. Where MetAssimulo 1.0 only simulated 1D 1H spectra of human urine, MetAssimulo 2.0 expands functionality to urine, blood, and cerebral spinal fluid (CSF), enhancing the realism of blood spectra by incorporating a broad protein background. This enhancement enables a closer approximation to real blood spectra, achieving a Pearson correlation of approximately 0.82. Moreover, this tool now includes simulation capabilities for 2D J-resolved (J-Res) and Correlation Spectroscopy (COSY) spectra, significantly broadening its utility in complex mixture analysis. MetAssimulo 2.0 simulates both single, and groups, of spectra with both discrete (case-control, e.g. heart transplant vs healthy) and continuous (e.g. BMI) outcomes and includes inter-metabolite correlations. It thus supports a range of experimental designs and demonstrating associations between metabolite profiles and biomedical responses.By enhancing NMR spectral simulations, MetAssimulo 2.0 is well positioned to support and enhance research at the intersection of deep learning and metabolomics.
    AVAILABILITY AND IMPLEMENTATION: The code and the detailed instruction/tutorial for MetAssimulo 2.0 is available at https://github.com/yanyan5420/MetAssimulo_2.git The relevant NMR spectra for metabolites are deposited in MetaboLights with accession number MTBLS12081.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btaf045
  22. J Chromatogr A. 2025 Jan 21. pii: S0021-9673(25)00060-3. [Epub ahead of print]1743 465711
    Method development and validation for the simultaneous determination of 21 antiviral drugs by ultra-high performance liquid chromatography-tandem mass spectrometry in chicken muscle and liver.
    Ana Castiñeira-Landeira, Samantha Sasse, Melissa Broeren, Saskia S Sterk, Ane Arrizabalaga-Larrañaga.
      The recent unauthorization of antiviral drugs in food-producing animals according to Commission Delegated Regulation (EU) 2022/1644 have increased the need for food control laboratories to develop analytical methods and perform official controls. In this work, a simple and fast analytical methodology was developed for the simultaneous determination of 21 antiviral drugs in chicken muscle and liver by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved by an HILIC BEH amide column; followed by detection with a electrospray ionization source in positive and negative modes. Based on extraction efficiencies, critical parameters affecting sample treatment were optimized including the evaporation and clean up steps to extract the largest number of antiviral drugs and reduce interferences. The method was validated according to Commission Implementing Regulation (EU) 2021/808 in chicken muscle and liver. Most compounds showed a linearity of R2>0.9800, while decision limits were between 0.18 and 7.05 μg kg-1 and 0.19 and 36 μg kg-1 for chicken muscle and liver, respectively. Trueness and within-lab reproducibility were determined at three levels (n = 7) and the results showed values ranging from 81 to 133 % and 4.2-57 % for chicken muscle, and 71-136 % and 4.6-106 % for chicken liver, respectively. The applicability of the developed method was demonstrated by the analysis real samples. 20 samples from the National Residue Control Plan in the Netherlands were analyzed and although none of targeted compounds were detected it is important to continue the analysis of larger set of samples to address any possible food safety risks.
    Keywords:  Control monitoring; Food safety; Hilic chromatography; LC-MS/MS; Residues; Target analysis
    DOI:  https://doi.org/10.1016/j.chroma.2025.465711
  23. Antioxidants (Basel). 2024 Dec 27. pii: 20. [Epub ahead of print]14(1):
    Direct Determination of Flavanone Isomers in Citrus Juice by Paper Spray Tandem Mass Spectrometry.
    Lucia Bartella, Fabio Mazzotti, Ilaria Santoro, Ines Rosita Talarico, Leonardo Di Donna.
      A novel and efficient analytical protocol based on paper spray tandem mass spectrometry was developed for the determination of isomeric O-glycoside flavanones in citrus juices and beverages. This approach significantly reduces sample preparation time and solvent consumption compared to traditional chromatographic techniques. By exploiting the unique fragmentation patterns of these compounds, accurate quantification of both diglycosides and their individual isomers (neohesperidoside and rutinose derivatives) was achieved. The method demonstrated excellent analytical performance, with high accuracy, selectivity, and reproducibility. The impact of matrix effects was mitigated through the construction of ratio calibration curves, ensuring reliable quantification in complex matrices. Finally, a simple DPPH experiment to assay the antioxidant activity of each single positional isomer was performed, indicating the superior ability of neohesperidose conjugates. This simplified method offers a valuable tool for quality control, authenticity assessment and the study of health benefits associated with citrus consumption.
    Keywords:  antioxidant compounds; citrus juices; flavanone isomers; paper spray ionization; tandem mass spectrometry
    DOI:  https://doi.org/10.3390/antiox14010020
  24. Sci Data. 2025 Jan 25. 12(1): 150
    Multidimensional library for the improved identification of per- and polyfluoroalkyl substances (PFAS).
    Kara M Joseph, Anna K Boatman, James N Dodds, Kaylie I Kirkwood-Donelson, Jack P Ryan, Jian Zhang, Paul A Thiessen, Evan E Bolton, Alan Valdiviezo, Yelena Sapozhnikova, Ivan Rusyn, Emma L Schymanski, Erin S Baker.
      As the occurrence of human diseases and conditions increase, questions continue to arise about their linkages to chemical exposure, especially for per-and polyfluoroalkyl substances (PFAS). Currently, many chemicals of concern have limited experimental information available for their use in analytical assessments. Here, we aim to increase this knowledge by providing the scientific community with multidimensional characteristics for 175 PFAS and their resulting 281 ion types. Using a platform coupling reversed-phase liquid chromatography (RPLC), electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), drift tube ion mobility spectrometry (IMS), and mass spectrometry (MS), the retention times, collision cross section (CCS) values, and m/z ratios were determined for all analytes and assembled into an openly available multidimensional dataset. This information will provide the scientific community with essential characteristics to expand analytical assessments of PFAS and augment machine learning training sets for discovering new PFAS.
    DOI:  https://doi.org/10.1038/s41597-024-04363-0
  25. Methods Mol Biol. 2025 ;2895 271-297
    Sample Preparation of Caenorhabditis elegans for GC-MS-Based Metabolomics in Toxicity Assessment.
    Jesus G Arellano Spadaro, Marjolein Wildwater, Henrie Korthout, Hye Kyong Kim.
      The nematode Caenorhabditis elegans, widely recognized as a model organism due to its ease of breeding and well-characterized genomes, boasts complete digestive, reproductive, and endocrine systems, as well as conserved signaling pathways shared with mammals. It has become an invaluable resource for metabolomics research, particularly in examining responses to chemical or environmental factors and toxicity assessments. In this article, we provide detailed, step-by-step protocols for cultivating C. elegans and conducting metabolomics analyses, specifically focusing on sample preparation for GC-MS analysis in response to toxic compound treatments. We highlight the critical choice of extraction solvent, introducing two representative systems for extracting metabolites from C. elegans.
    Keywords:  Caenorhabditis elegans; Extraction; Extraction solvent; GC-MS analysis; Metabolomics; Toxicity assessment
    DOI:  https://doi.org/10.1007/978-1-0716-4350-1_18
  26. Methods Mol Biol. 2025 ;2895 245-257
    Identification of Primary Metabolites in 1H NMR-Based Metabolomics of Plants.
    Hye Kyong Kim, Young Hae Choi.
      Identifying metabolites in NMR-based plant metabolomics is challenging due to the complexity of plant metabolites. This complexity stems from the abundance and diverse chemical properties of compounds, which vary in concentration across plant specimens. Additionally, the lack of automated identification software complicates the analysis process. Primary metabolites such as amino acids and sugars are widespread in plants, yet their identification is not straightforward due to various stereoisomeric forms and dynamic equilibria. Our protocol offers a manual approach to identify these metabolites, particularly focusing on amino acids and sugars. Through step-by-step guidance, we aim to empower researchers to navigate plant metabolomics complexities effectively.
    Keywords:  Amino acid; Identification; NMR spectroscopy; Plant metabolomics; Primary metabolites
    DOI:  https://doi.org/10.1007/978-1-0716-4350-1_16
  27. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jan 18. pii: S1570-0232(25)00024-8. [Epub ahead of print]1253 124472
    Development of a novel HPLC-HRMS method for quantitative analysis of resorcinol in urine: Application to hairdressers' occupational exposure.
    Amandine Cambrai-Erb, Flavien Denis, Romain Pons, Anca Radauceanu, Sophie Ndaw, Nathalie Grova.
      Resorcinol is a widespread substance used in a large variety of manufacturing industries, including cosmetics, with endocrine-disrupting activity on the thyroid function. The aim of the present study was to develop and validate a sensitive, selective and robust method to quantify resorcinol in urine and thereby assess hairdressers' occupational exposure. As resorcinol is mainly excreted in urine as glucuronide or sulfate forms, the first step consisted in hydrolyzing urine samples with a β-glucuronidase-arylsulfatase enzyme for 16 h. Then, after cleaning with a supported-liquid extraction cartridge, the samples were derivatized with dansyl chloride to improve signal and signal-to-noise ratio. Analysis was carried out using an accurate high-resolution liquid chromatography-mass spectrometry instrument on a Kinetex Biphenyl analytical column. Particular attention was paid to the chromatographic separation of resorcinol from its two isomers, catechol and hydroquinone, also present in urine. Acquisition was performed in positive ESI mode, at m/z 577.14615 for dansylated resorcinol and m/z 581.17126 for dansylated resorcinol-d4, with respective retention times of 8.63 and 8.60 min. The method passed all the performance tests included in the validation process. The lowest limit of quantification (LLOQ) was 0.3 µg/L resorcinol, which was sufficient to quantify resorcinol in all samples tested. The calibration curves were linear from LLOQ to 2000 µg/L, with coefficients of determination R2 ranging from 99.82 % to 100 %. The method was accurate, reaching 95.6 to 101.7 % of target intraday and 99.8 to 105.0 % interday, and precise with RSDs between 0.88 and 1.99 % intraday and with RSDs between 1.75 and 8.65 % in interday assessments. It also proved robust, with a matrix effect of 8.25 %. Resorcinol stability was determined by studying long-term stability at -20 °C for sample storage up to 6 months, short-term stability (at + 20 °C and + 4°C for possible short-term storage), freeze-thaw cycles, and post derivatization stability. This method was successfully applied on samples from 17 women working as hairdressers. Urinary resorcinol concentrations ranged from 2 µg/L to 1824 µg/L (6 to 4475 µg/g creatinine) and were in line with those reported in the literature.
    Keywords:  HPLC-HRMS; Hairdresser; Occupational exposure; Resorcinol; Urine
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124472
  28. J Am Soc Mass Spectrom. 2025 Jan 27.
    A Fast-Pass, Desorption Electrospray Ionization Mass Spectrometry Strategy for Untargeted Metabolic Phenotyping.
    Hawkins S Shepard, Jody C May, Baltazar E Zuniga, Joshua P Abraham, Brian F Pfleger, Jamey D Young, John A McLean.
      Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) provides direct analytical readouts of small molecules that can be used to characterize the metabolic phenotypes of genetically engineered bacteria. In an effort to accelerate the time frame associated with the screening of mutant libraries, we have developed a high-throughput DESI-MSI analytical workflow implementing a single raster line-scan strategy that facilitates the collection of location-resolved molecular information from engineered strains on a subminute time scale. Evaluation of this "Fast-Pass" DESI-MSI phenotyping workflow on analytical standards demonstrated the capability of acquiring full metabolic profiling information with a throughput of ∼40 s per sample. This Fast-Pass strategy was implemented in the analysis of genetically edited Escherichia coli strains that have been engineered to produce various free-fatty acids (FFAs) for applications relevant to biofuels. Due to the untargeted nature of DESI-MSI, the investigation of these strains yielded molecular information for both global metabolites and targeted detection of accumulated bioproducts, allowing simultaneous readouts of strain-specific chemical profiles and comparative measurements of FFA production levels.
    Keywords:  DESI-MS; lipidomics; mass spectrometry imaging; metabolomics; molecular engineering; synthetic biology
    DOI:  https://doi.org/10.1021/jasms.4c00459
  29. Food Chem X. 2025 Jan;25 102167
    Development and application of an efficient, accurate, and environmentally friendly liquid chromatography-tandem mass spectrometry method for the determination of five Alternaria toxins in wheat.
    Huiyuan Lang, Zengwang Guo, Yu Wu, Li Li, Hongmei Liu, Lianzhou Jiang, Songxue Wang, Jin Ye.
      The contamination of Alternaria toxins poses a potential risk to human health. This study developed a rapid, efficient, and environmentally friendly method for the simultaneous determination of five types of Alternaria toxins in wheat using high-precision and stable isotope liquid chromatography tandem mass spectrometry. The comparison between dilution method and solid-phase extraction method shows that the former achieves satisfactory results with a simple and convenient sample purification method. The quantitative limit range is 0.88 to 1.68 μg/kg. The recoveries are between 81.40% and 102.68%, with RSD less than 11.95%. The method was used to analyze 60 samples from the main wheat producing areas in China. The results showed that Tenuzonic acid had the highest detection rate (100%), followed by Tentoxin (95%), Alternariol (66.67%), and Alternariol monomethyl ether (53.33%). There is a certain pollution risk that needs to be taken seriously and monitoring should be strengthened.
    Keywords:  Alternaria toxins; Contamination assessment; Dilution method; LC-MS/MS; Wheat
    DOI:  https://doi.org/10.1016/j.fochx.2025.102167
  30. Nat Commun. 2025 Jan 27. 16(1): 1065
    msiFlow: automated workflows for reproducible and scalable multimodal mass spectrometry imaging and microscopy data analysis.
    Philippa Spangenberg, Sebastian Bessler, Lars Widera, Jenny Bottek, Mathis Richter, Stephanie Thiebes, Devon Siemes, Sascha D Krauß, Lukasz G Migas, Siva Swapna Kasarla, Prasad Phapale, Jens Kleesiek, Dagmar Führer, Lars C Moeller, Heike Heuer, Raf Van de Plas, Matthias Gunzer, Oliver Soehnlein, Jens Soltwisch, Olga Shevchuk, Klaus Dreisewerd, Daniel R Engel.
      Multimodal imaging by matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI MSI) and microscopy holds potential for understanding pathological mechanisms by mapping molecular signatures from the tissue microenvironment to specific cell populations. However, existing software solutions for MALDI MSI data analysis are incomplete, require programming skills and contain laborious manual steps, hindering broadly applicable, reproducible, and high-throughput analysis to generate impactful biological discoveries. Here, we present msiFlow, an accessible open-source, platform-independent and vendor-neutral software for end-to-end, high-throughput, transparent and reproducible analysis of multimodal imaging data. msiFlow integrates all necessary steps from raw data import to analytical visualisation along with state-of-the-art and self-developed algorithms into automated workflows. Using msiFlow, we unravel the molecular heterogeneity of leukocytes in infected tissues by spatial regulation of ether-linked phospholipids containing arachidonic acid. We anticipate that msiFlow will facilitate the broad applicability of MSI in multimodal imaging to uncover context-dependent cellular regulations in disease states.
    DOI:  https://doi.org/10.1038/s41467-024-55306-7
  31. Bio Protoc. 2025 Jan 20. 15(2): e5168
    Using DIMet for Differential Analysis of Labeled Metabolomics Data: A Step-by-step Guide Showcasing the Glioblastoma Metabolism.
    Johanna Galvis, Joris Guyon, Thomas Daubon, Macha Nikolski.
      Stable-isotope resolved metabolomics (SIRM) is a powerful approach for characterizing metabolic states in cells and organisms. By incorporating isotopes, such as 13C, into substrates, researchers can trace reaction rates across specific metabolic pathways. Integrating metabolomics data with gene expression profiles further enriches the analysis, as we demonstrated in our prior study on glioblastoma metabolic symbiosis. However, the bioinformatics tools for analyzing tracer metabolomics data have been limited. In this protocol, we encourage the researchers to use SIRM and transcriptomics data and to perform the downstream analysis using our software tool DIMet. Indeed, DIMet is the first comprehensive tool designed for the differential analysis of tracer metabolomics data, alongside its integration with transcriptomics data. DIMet facilitates the analysis of stable-isotope labeling and metabolic abundances, offering a streamlined approach to infer metabolic changes without requiring complex flux analysis. Its pathway-based "metabologram" visualizations effectively integrate metabolomics and transcriptomics data, offering a versatile platform capable of analyzing corrected tracer datasets across diverse systems, organisms, and isotopes. We provide detailed steps for sample preparation and data analysis using DIMet through its intuitive, web-based Galaxy interface. To showcase DIMet's capabilities, we analyzed LDHA/B knockout glioblastoma cell lines compared to controls. Accessible to all researchers through Galaxy, DIMet is free, user-friendly, and open source, making it a valuable resource for advancing metabolic research. Key features • Glioblastoma tumor spheroids in vitro replicate tumors' three-dimensional structure and natural nutrient, metabolite, and gas gradients, providing a more realistic model of tumor biology. • Joint analysis of tracer metabolomics and transcriptomics datasets provides deeper insights into the metabolic states of cells. • DIMet is a web-based tool for differential analysis and seamless integration of metabolomics and transcriptomics data, making it accessible and user-friendly. • DIMet enables researchers to infer metabolic changes, offering intuitive and visually appealing "metabologram" outputs, surpassing conventional visual representations commonly used in the field.
    Keywords:  Bioinformatics; Data integration; Differential analysis; Glioblastoma; Metabolomics; Transcriptomics
    DOI:  https://doi.org/10.21769/BioProtoc.5168
  32. Rapid Commun Mass Spectrom. 2025 May 15. 39(9): e9994
    Fentanyl and Fentanyl Analog Screening Using ASAP-MS With LiveID Confirmation.
    Karen A Reyes Monroy, Rachel Koerber, Guido F Verbeck.
       RATIONALE: Fentanyl and fentanyl analogs continue to pose a serious threat to the public health. The vast number of fentanyl analogs emerging on the black-market call for optimized analytical methods for the detection, analysis, and characterization of these extremely dangerous drugs.
    METHODS: Atmospheric pressure solids analysis probe (ASAP) mass spectrometry was used for the rapid analysis of 250 synthetic opioid standards, including 211 fentanyl analogs, 32 non-fentanyl related opioids, and 8 fentanyl precursors. Four cone voltages (5, 15, 35 and 50 V) were used to obtain fingerprint data for each opioid reference sample.
    RESULTS: The generated and processed mass spectra of the 250 synthetic opioids analyzed was utilized to create an ASAP+ database that contains the largest compendium of mass spectra for fentanyl analogs. The built library was integrated into LiveID software, enabling real-time analyte identification. The efficacy of the software's ability to identify fentanyl analogs in a sample utilizing the spectral library was tested by analyzing five blind and four reaction mixtures. The correct identity of these nine samples was all within the top three ranked matches.
    CONCLUSION: We demonstrate how the RADIAN ASAP, alongside a real-time sample recognition software, can be utilized as a presumptive tool for the screening of fentanyl analogs within samples in question, making it a promising alternative to some of the most commonly used analytical screening techniques.
    Keywords:  LiveID software; atmospheric pressure solids analysis probe (ASAP); fentanyl; fentanyl analogs; fentanyl database; fentanyl fragmentation pathways; mass spectrometry; novel psychoactive substances (NPS)
    DOI:  https://doi.org/10.1002/rcm.9994
  33. Int J Mol Sci. 2025 Jan 09. pii: 489. [Epub ahead of print]26(2):
    Optimization and Validation of an Ultra-Performance Liquid Chromatography with Quadrupole Detector Mass Spectrometry Quantification Method for the Simultaneous Detection of Tazarotene and Tazarotenic Acid in Porcine Skin: An In Vitro Study.
    Helena Hamzehpour, Kristófer H Hauksson, Helgi Jónsson, Sveinbjorn Gizurarson, Bergthora S Snorradottir.
      Exploring tazarotene, a third-generation retinoid for potential hand osteoarthritis treatment, this study presents the development and validation of an ultra-performance liquid chromatography with quadrupole detector mass spectrometry (UPLC-QDa) method for the simultaneous quantification of tazarotene and tazarotenic acid, its active metabolite, in porcine skin. Method development involved a design-of-experiments approach for chromatographic optimization of gradient steepness, organic solvent volume, column temperature, capillary voltage, flow rate, and cone voltage. Central composite orthogonal design was used to optimize peak area, peak width, retention time, and resolution. Validation was performed in accordance with U.S. Food and Drug Administration guidelines. The method was linear over the concentration range of 0.4-18,750 ng/mL for tazarotene and 13.3-12,500 ng/mL for tazarotenic acid, with r2 values of ≥0.99. Chromatographic analysis demonstrated acceptable accuracy and precision (<15%), and stability tests confirmed the analytes' stability under various conditions. This validated method offers a reliable and accurate approach for the simultaneous analysis of tazarotene and tazarotenic acid, facilitating further research into their therapeutic applications for hand osteoarthritis.
    Keywords:  bioanalytical techniques; design of experiments (DoE); hand osteoarthritis; quadrupole detector mass spectrometry (QDa); tazarotene; tazarotenic acid; transdermal
    DOI:  https://doi.org/10.3390/ijms26020489
  34. Methods Mol Biol. 2025 ;2895 227-243
    Analysis and Identification of Polyphenols in Fragaria, Rubus, and Ribes Species by Nontargeted UHPLC-PDA-MS Metabolite Profiling.
    Simon D A Pont, J William Allwood.
      Profiling of secondary metabolites within Fragaria sp. (strawberry), Rubus sp. (raspberries and blackberries), and Ribes sp. (blackcurrants and redcurrants) is of key importance with respect to the nutritional (antioxidant) benefits associated with a range of polyphenol classes within such fruits and how they vary between growth environment and season, as well as their deployment as quantitative trait loci (QTL) within breeding populations. This chapter presents an ultra-high-performance liquid chromatography (UHPLC) approach applying aqueous functionalized C18 chromatography combined with mass spectrometry (MS) for the characterization and relative quantification of a wide range of polyphenols such as the pelargonidins, being rich in strawberry; cyanidins, procyanidins, and anthocyanins, being rich in raspberry; and delphinidins and prodelphinidins, being particularly rich in blackcurrant, as well as a range of colorless flavonoids, and amino and organic acids.
    Keywords:  Data-dependent analysis (DDA); Fragaria; Mass spectrometry (MS); Ribes; Rubus; Tandem MS (MS/MS); Ultra-high-performance liquid chromatography (UHPLC)
    DOI:  https://doi.org/10.1007/978-1-0716-4350-1_15
  35. Anal Chem. 2025 Jan 29.
    Stereoselective Reaction Enabling Simultaneous Analysis of Carbon-Carbon Double-Bond Configuration and the Position of Monounsaturated Fatty Acids through UHPLC-ESI-MRM-MS.
    Chunlin Yue, Lingyu Zhao, Zhenchao Long, Xuwei Song, Jinqi Yang, Yuan Cao, Yuanyuan Zhang, Yangyang Zhang, Zhenwen Zhao.
      Monounsaturated fatty acids (MUFA) are an important class of nutrients and are involved in lipid metabolism. The positions of the C=C bond and cis-trans isomerism have a significant influence on their physiological activity. However, simultaneously detecting these two structural properties has been challenging due to multiple isomers of MUFA. In this study, we developed a method that involved an N-aminophthalimide (PhthNH2) derivatization of the C=C bond in MUFA, followed by analysis using ultrahigh-performance liquid chromatography-electrospray ionization-multiple reaction monitoring mass spectrometry (UHPLC-ESI-MRM-MS) technology to achieve analysis of cis-trans isomers and positional isomers of the C=C bond. The derivatives of cis-trans isomers of the C=C bond were well separated in UHPLC, and their corresponding C=C bond positions were deduced from characteristic fragment ions in tandem mass spectrometry. With our method, we found MUFA with different double-bond positions and cis-trans isomers in several samples, including mouse kidney, butter, etc., achieving qualitative analysis and relative quantitation. This method is expected to be applied by more researchers in lipidomics studies in the future.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06262
  36. Adv Biotechnol (Singap). 2024 Mar 11. 2(1): 11
    Exploring the plant lipidome: techniques, challenges, and prospects.
    Hao-Zhuo Liu, Yong-Kang Li, Yi-Li Chen, Ying Zhou, Sunil Kumar Sahu, Ningjing Liu, Hao Wu, Guanghou Shui, Qinfang Chen, Nan Yao.
      Plant lipids are a diverse group of biomolecules that play essential roles in plant architecture, physiology, and signaling. To advance our understanding of plant biology and facilitate innovations in plant-based product development, we must have precise methods for the comprehensive analysis of plant lipids. Here, we present a comprehensive overview of current research investigating plant lipids, including their structures, metabolism, and functions. We explore major lipid classes, i.e. fatty acids, glyceroglycolipids, glycerophospholipids, sphingolipids, and phytosterols, and discuss their subcellular distributions. Furthermore, we emphasize the significance of lipidomics research techniques, particularly chromatography-mass spectrometry, for accurate lipid analysis. Special attention is given to lipids as crucial signal receptors and signaling molecules that influence plant growth and responses to environmental challenges. We address research challenges in lipidomics, such as in identifying and quantifying lipids, separating isomers, and avoiding batch effects and ion suppression. Finally, we delve into the practical applications of lipidomics, including its integration with other omics methodologies, lipid visualization, and innovative analytical approaches. This review thus provides valuable insights into the field of plant lipidomics and its potential contributions to plant biology.
    Keywords:  Biomolecules; Environmental stress; Lipidomics; Mass spectrometry; Plant lipids
    DOI:  https://doi.org/10.1007/s44307-024-00017-9
  37. Front Neurosci. 2024 ;18 1520982
    MetaboLINK is a novel algorithm for unveiling cell-specific metabolic pathways in longitudinal datasets.
    Jared Lichtarge, Gerarda Cappuccio, Soumya Pati, Alfred Kwabena Dei-Ampeh, Senghong Sing, LiHua Ma, Zhandong Liu, Mirjana Maletic-Savatic.
       Introduction: In the rapidly advancing field of 'omics research, there is an increasing demand for sophisticated bioinformatic tools to enable efficient and consistent data analysis. As biological datasets, particularly metabolomics, become larger and more complex, innovative strategies are essential for deciphering the intricate molecular and cellular networks.
    Methods: We introduce a pioneering analytical approach that combines Principal Component Analysis (PCA) with Graphical Lasso (GLASSO). This method is designed to reduce the dimensionality of large datasets while preserving significant variance. For the first time, we applied the PCA-GLASSO algorithm (i.e., MetaboLINK) to metabolomics data derived from Nuclear Magnetic Resonance (NMR) spectroscopy performed on neural cells at various developmental stages, from human embryonic stem cells to neurons.
    Results: The MetaboLINK analysis of longitudinal metabolomics data has revealed distinct pathways related to amino acids, lipids, and energy metabolism, uniquely associated with specific cell progenies. These findings suggest that different metabolic pathways play a critical role at different stages of cellular development, each contributing to diverse cellular functions.
    Discussion: Our study demonstrates the efficacy of the MetaboLINK approach in analyzing NMR-based longitudinal metabolomic datasets, highlighting key metabolic shifts during cellular transitions. We share the methodology and the code to advance general 'omics research, providing a powerful tool for dissecting large datasets in neurobiology and other fields.
    Keywords:  Glasso; MetaboLINK; PCA; embryonic bodies; hESC; metabolome; neuroprogenitors; rosettes
    DOI:  https://doi.org/10.3389/fnins.2024.1520982
  38. Rapid Commun Mass Spectrom. 2025 Jan 31. e9996
    Unveiling Impurity Profiling of Synthetic Pathways of Organophosphorus Chlorpyrifos Through LC-HRMS Metabolomics-Based Approaches.
    C Orlandi, G Delaporte, C Albaret, E Joubert, A Bossée, L Debrauwer, E L Jamin.
      Sourcing in chemical forensic science refers to the attribution of a sample to a specific source using a characteristic signature. It relies on the identification of chemical attribution signatures (CAS), including chemical markers such as residual synthetic precursors, impurities, reaction by-products and degradation products, or even metabolites. Undertaking CAS for chemical threat agents (CTA) can be used to provide an evidentiary link between the use of a given chemical and its precursor(s) to support forensic investigations. Organophosphorus compounds, a class of nerve agents, can be produced by different, more or less complex synthesis routes that can lead to specific CAS. Chlorpyrifos (CPF), an organophosphorus pesticide, was selected as model compound. To assess the specificity of impurity markers originated from a chemical synthesis, untargeted fingerprints of crude CPF from different synthesis pathways were analyzed as a first use-case using metabolomics-based trace discovery strategies. Seven different CPF synthesis routes were considered, and their crude mixtures were analyzed with a minimal sample preparation. Analyses were performed on a trapped ion mobility spectrometry (TIMS) coupled to liquid chromatography (LC) and high-resolution mass spectrometry (HRMS). Chemometrics analyses were conducted with multivariate methods to extract discriminating features (i.e., relevant impurities), annotate, and identify them. Then, unknown samples were analyzed in blind conditions without any information of the synthesis pathway employed. The aim is to validate the methodology seeking some discriminating impurities identified in the first section to attribute and classify them according to the synthesis route.
    Keywords:  chemical attribution signature; chemical threat agents; chlorpyrifos; forensic; high‐resolution mass spectrometry; metabolomics; organophosphorus chemical
    DOI:  https://doi.org/10.1002/rcm.9996
  39. Anal Chem. 2025 Jan 27.
    Two-Dimensional Liquid Chromatography-Isotope Ratio Mass Spectrometry Opens New Possibilities in the Field of Compound-Specific Stable Isotope Analysis.
    Sarah P Rockel, Robert G H Marks, Klaus Kerpen, Maik A Jochmann, Torsten C Schmidt.
      Compound-specific stable isotope analysis (CSIA) using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) is a powerful tool for determining the isotopic composition of carbon in analytes from complex mixtures. However, LC-IRMS methods are constrained to fully aqueous eluents. Previous efforts to overcome this limitation were unsuccessful, as the use of organic eluents in LC-IRMS was deemed impossible. In our study, we developed a two-dimensional (2D) LC-IRMS method that, for the first time, enables the use of organic-containing eluents in an LC-IRMS setting. Initial experiments with caffeine were performed using a sample loop modulator with 20% methanol in the mobile phase of the first dimension, while separating the organic fraction from the analyte in the second dimension. Comparing results with one-dimensional (1D) LC-IRMS methods showed high precision with δ13C values in 2D measurements (-34.98 ± 0.04 ‰) closely matching 1D results (-34.95 ± 0.12 ‰). In the next step, incorporation of an at-column dilution (ACD) modulator allowed for the successful use of methanol concentrations up to 40% in the first dimension, with the ACD modulator effectively mitigating both peak fronting and carbon background interference, without losing any precision or accuracy of the measurements (δ13CCaffeine = -34.92 ± 0.03 ‰). All developed methods showed a method detection limit lower than 5 mg of carbon L-1 (mgC L-1), which is a major improvement compared with previous studies on caffeine analysis with LC-IRMS. This proof-of-concept study on 2D-LC-IRMS opens vast new possibilities for future CSIA research across diverse fields, including environmental science, pharmaceuticals, and food chemistry.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05956
  40. J Asian Nat Prod Res. 2025 Jan 31. 1-14
    Analytical method development and validation for monosaccharide profiling in Lignosus rhinocerotis using rP-HPLC.
    Solehah Mohd Rosdan Bushra, Ruzilawati Abu Bakar, Asma Abdullah Nurul.
      Lignosus rhinocerotis is rich in polysaccharide with diverse -bioactivities. This study developed a pre-column derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method for analyzing monosaccharides in Lignosus rhinocerotis polysaccharides (LRP). LRP underwent hydrolysis, derivatization, and separation on a Cosmosil 5C18-MS-II column at 254 nm. Baseline separation of eight standard monosaccharides was achieved within 45 min. Calibration curves, precision, and accuracy were validated. Quantitative analysis revealed LRP as a heteropolysaccharide containing mannose, ribose, rhamnose, glucose, galactose, xylose, and arabinose, with 100.28-111.02% recovery. This optimized RP-HPLC offers a simple, reproducible, and accurate tool for LRP monosaccharides analysis, facilitating in understanding its structure-function relationship.
    Keywords:  HPLC; Lignosus rhinocerotis; derivatization; monosaccharide; polysaccharide
    DOI:  https://doi.org/10.1080/10286020.2025.2453852
  41. Anal Chem. 2025 Jan 27.
    QuanFormer: A Transformer-Based Precise Peak Detection and Quantification Tool in LC-MS-Based Metabolomics.
    Zhengyi Zhang, Huan Yang, Yanyi Wang, Lei Zhang, Shu-Hai Lin.
      In metabolomic analysis based on liquid chromatography coupled with mass spectrometry, detecting and quantifying intricate objects is a massive job. Current peak picking methods still cause high rates of incorrectly picked peaks to influence the reliability and reproducibility of results. To address these challenges, we developed QuanFormer, a deep learning method based on object detection designed to accurately quantify peak signals. Our algorithm combines the feature extraction capabilities of convolutional neural networks (CNNs) with the global computation capability of Transformer architecture. Data training in QuanFormer by using nearly 20,000 annotated regions-of-interest (ROIs) ensures unique prediction via bipartite matching, achieving 96.5% of the average precision value on the test set. Even without retraining, QuanFormer achieves over 90% accuracy in distinguishing true from false peaks. Performance was further analyzed using visualization techniques applied to the encoder and decoder layers. We also demonstrated that QuanFormer could correct retention time shifts for peak alignment and generally surpass the existing methods, including MZmine 3 and PeakDetective, to obtain a larger number of picked peaks and higher accurate quantification. Finally, we also carried out metabolomic analysis in a clinical cohort of breast cancer patients and utilized QuanFormer to detect and quantify the potential biomarkers. QuanFormer is open-source and available at https://github.com/LinShuhaiLAB/QuanFormer.
    DOI:  https://doi.org/10.1021/acs.analchem.4c04531
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