bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2026–03–15
fifty-one papers selected by
Sofia Costa, Matterworks
  1. Investigating Variability in Metabolomics: A Comparative Study of Analytical Platforms and Blood Matrices Using HPLC-HRMS.
  2. Comprehensive lipidomic characterization of human blood by high-throughput UHPSFC/MS using bioinert column and modified MTBE extraction.
  3. An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of linezolid in human serum and plasma.
  4. First validated liquid chromatography-tandem mass spectrometry method for simultaneous quantification of propranolol and 4-hydroxypropranolol in pig plasma and dried blood spots and its application to a pharmacokinetic study.
  5. Polar microcystins or arginine methyl ester can serve as sensitive reference materials for system suitability tests in untargeted metabolomics using reversed-phase LC-MS: A case study.
  6. [Simultaneous determination of selinexor， posaconazole， venetoclax， and voriconazole in human plasma using ultra-high performance liquid chromatography-tandem mass spectrometry].
  7. Detection and quantification of ezetimibe and its major glucuronide in patients with hepatic impairment via liquid chromatography-tandem mass spectrometry.
  8. Quantitative Determination Method for Residual Colistin A/B in Edible Meats and Milk by Liquid Chromatography-Tandem Mass Spectrometry.
  9. [Determination of 30 homologues of phosphatidylcholines and lysophosphatidylcholines in human serum by liquid chromatography-tandem mass spectrometry and their correlation analysis with coronary artery disease].
  10. S'Wipe: user-friendly stool collection for high-throughput gut metabolomics and multi-omics.
  11. Development and Validation of a UPLC-MS/MS Method for the Determination of Apalutamide in Human Plasma and Its Application in a Bioequivalence Study.
  12. Simple and sensitive cellulose paper-based microextraction of 25 steroid esters from human serum/plasma for doping control purposes.
  13. An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of 17-hydroxyprogesterone in human serum and plasma.
  14. [Performance validation and consistency assessment of three liquid chromatography-tandem mass spectrometry methods for detecting fat-soluble vitamins].
  15. [Establishment and international comparison of reference methods for glycated hemoglobin].
  16. Performance evaluation of automated magnetic beads extraction method for the measurement of 23 plasma steroids using dual liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
  17. [Establishment and application of a knowledge-directed pseudo-targeted analytical method for diabetic retinopathy].
  18. Differentiation of cannabinoid isomers via Cu-mediated molecular ion formation and electrospray ionization-tandem mass spectrometry.
  19. Preparation of styrene-maleic anhydride copolymer nanobrush chromatographic stationary phase and its application in phospholipid analysis of exosomes.
  20. Validated LC-MS/MS method for the quantification of glyphosate and its metabolites in human serum: A biomonitoring study in farming intensive regions of the Western Cape, South Africa.
  21. Simultaneous quantification of venetoclax, busulfan and voriconazole in plasma by LC-MS/MS: Clinical applications in patients with acute myeloid leukemia.
  22. Shared-autosampler parallel LC-MS/MS platform for high-throughput multi-analyte quantification in complex matrices.
  23. Rapid Analysis of Phytic Acid by Paper Spray Mass Spectrometry.
  24. Chiral derivatization reagents for enantioselective LC-MS/MS analysis of amino acids with analyte-specific fragmentation.
  25. [Rapid determination of venetoclax in plasma by ultra performance liquid chromatography-tandem mass spectrometry].
  26. Multiplexed LC-MS analysis reveals novel insights into grapevine defense mechanisms by expanding metabolome coverage.
  27. Enantioselective Determination of Carnitine Enantiomers in Food and Supplement Samples by Chiral Liquid Chromatography-Tandem Mass Spectrometry.
  28. Towards large-scale mass spectrometry-based omics for clinical applications.
  29. Ultra-Selective Ultra-High-Performance Liquid Chromatography-Triple-Stage Mass Spectrometry Assay for Quantification of Pyraclostrobin in Zebrafish Liver Tissues Via Multistage Fragmentation to Enhance Sensitivity.
  30. Resolving sn-Positional Isomers of Docosahexaenoic Acid-Bound Phospholipids in the Mouse Brain by Cyclic Ion Mobility Mass Spectrometry Imaging.
  31. [Development of a liquid chromatography-tandem mass spectrometry method for steroid hormone detection during pregnancy and consistency analysis with chemiluminescence immunoassay].
  32. Changes in Metabolism and Lipid Composition with Nitrogen Starvation and Recovery in a New Productive Strain of Neochlorella semenenkoi Using N15-Isotopic Labeling and HRMS.
  33. Resolving lipid isomers up to the double bond position level using reversed-phase chromatography, EAD fragmentation, and high-resolution mass spectrometry: Application to pancreatic cancer.
  34. Faster, simpler, and more precise calibration curves: Expanding the scope of continuous calibration.
  35. [Application of accuracy profile-based evaluation in the development of quantitative methods for urinary phenol detection].
  36. Bioanalysis of amphetamines in alternative matrices using a sensitive and validated liquid chromatography-tandem mass spectrometry method and its application to real samples.
  37. [Application of chromatography-mass spectrometry in the clinical analysis of multiple sclerosis].
  38. [Pharmacokinetics of three pairs isomers of Caraway in Beagle dogs based on UPLC-MS/MS].
  39. Hydrophobic covalent organic framework as a solid-phase microextraction coating for high-throughput quantification of endocannabinoids in Parkinson's disease rat plasma.
  40. [Determination of vitamin A and vitamin E in human tear by liquid chromatography-tandem mass spectrometry and analysis of their relationship with dry eye].
  41. Optimization of an Extraction Protocol for Untargeted Metabolomics of Vitis vinifera L. Leaves.
  42. Development and validation of an ultra-sensitive UPLC-MS/MS method for quantification of monomethyl auristatin E in cynomolgus monkey plasma: application to pharmacokinetic study.
  43. Efficient and Simultaneous LC-MS/MS Detection of N-Nitroso and Di-Nitroso Leucovorin in Leucovorin Drug Products: Method Development and Validation.
  44. N-glycan mass spectrometry imaging - a valuable tool for biomarker discovery and validation.
  45. Simultaneous Quantification of Eight Chiral and Achiral Components in Notopterygii Rhizoma et Radix Extract and Rat Plasma Based on Chiral Stationary Phase-HPLC-MS/MS.
  46. Simultaneous Analysis of Biomarkers in Human Hair for Evaluating Chronic Tobacco Smoke Exposure and Stress/Relaxation Using Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry.
  47. An on-tissue parallel d0/d20-meladrazine derivatization approach for fatty acid peroxidation products imaging in adult zebrafish.
  48. Separating O-desmethylvenlafaxine and tramadol enantiomers using two-dimensional chiral LC × DMS mass spectrometry.
  49. Feed injection-enabled reversed phase liquid chromatography for simplified analysis of lipophilic drugs and formulations.
  50. [Analysis of urine biomarkers in urothelial carcinoma based on untargeted metabolomics].
  51. Targeted serum metabolomics reveals novel metabolic associations between fatty acid and kynurenine metabolism in nonalcoholic fatty liver.
  1. Molecules. 2026 Feb 28. pii: 814. [Epub ahead of print]31(5):
    Investigating Variability in Metabolomics: A Comparative Study of Analytical Platforms and Blood Matrices Using HPLC-HRMS.
    Giulia Guerra, Alessio Polymeropoulos, Elisabetta Venturelli, Veronica Huber, Francesco Segrado, Daniele Morelli, Sabina Sieri.
      Untargeted metabolomics faces significant challenges in standardization due to variability introduced by sample preparation and analytical workflows. We systematically evaluated the impact of biological matrices, extraction protocols, and chromatographic configurations to establish a mechanism-informed framework aimed at improving reproducibility in large-scale clinical and epidemiological studies. Three extraction protocols were compared using an in-house pooled heparin plasma: monophasic protein precipitation with isopropanol (IPA), methanol:acetonitrile (MeOH:ACN), and a modified Matyash biphasic method. The most reproducible protocol was then applied to four blood matrices. Samples were analysed using untargeted metabolomics on hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) HPLC columns, with mass spectrometry data processed using Compound Discoverer. Both IPA and MeOH:ACN extractions achieved over 80% of features with coefficient of variation (CV%) ≤ 30% for both RP and HILIC, whereas the Matyash method showed higher variability, with a larger proportion of metabolites exhibiting CV% > 30%. Across matrices, RP chromatography detected over 80% of metabolites with CV% < 30%, while HILIC showed higher variability, with at least 20% of metabolites above this threshold. Among matrices, serum and heparin plasma outperformed EDTA and citrate in reproducibility. We propose a standardized workflow in which monophasic extractions combined with RP chromatography maximize reproducibility and metabolite coverage, minimizing methodological artefacts and providing a reliable framework for robust biological discovery in large-scale untargeted metabolomics studies.
    Keywords:  blood matrix; data quality; human plasma; metabolomics; validation
    DOI:  https://doi.org/10.3390/molecules31050814
  2. Anal Chim Acta. 2026 May 01. pii: S0003-2670(26)00213-8. [Epub ahead of print]1397 345263
    Comprehensive lipidomic characterization of human blood by high-throughput UHPSFC/MS using bioinert column and modified MTBE extraction.
    Petra Peroutková, Robert Jirásko, Anna Taylor, Michal Holčapek, Ondřej Peterka.
       BACKGROUND: Supercritical fluid chromatography is traditionally employed for nonpolar and moderately polar analytes, while the analysis of ionic compounds remains a recognized limitation of the technique. Moreover, some polar lipids may contain a chromatographically challenging ionic group, which can interact with the metal surfaces of the instrument and column, resulting in poor peak shape and loss of sensitivity. Here, we introduce a novel ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method using a bioinert column, enabling the separation of lipids with a broad polarity range from nonpolar to ionic species.
    RESULTS: The UHPSFC/MS method was optimized using 79 lipid species across 41 lipid subclasses, achieving a total run time of 7.5 min, including the column equilibration. The comparison of the separation with conventional and bioinert columns revealed a substantial improvement in peak shapes for ionic lipid classes, such as PS, LPS, PA, LPA, CerP, and SPBP. Additionally, we introduce a combination of the modified chloroform-free extraction followed by a hexane elimination step. The optimized methodology was applied for the untargeted analysis of human plasma and erythrocyte-rich fraction to achieve highly confident identification of 657 lipid species across 37 lipid subclasses in human blood. The method follows the recommendations for validation of (bio)analytical methods, and its accuracy was confirmed by quantitative analysis of the reference material NIST SRM 1950, with the determined concentrations in agreement with the consensus values from ring trials.
    SIGNIFICANCE: The current methodology represents a novel high-throughput and comprehensive quantitative lipidomic method for biological samples. The modified MTBE extraction enhances workflow efficiency by reducing concentrations of nonpolar lipids, which enables injection of more concentrated lipid extracts while minimizes ion source contamination. Moreover, the findings highlight the potential for the development of bioinert components specifically designed for SFC platforms, enabling broader applicability of the technique.
    Keywords:  Bioinert column; Human blood; Lipid extraction; Lipidomics; Mass spectrometry; Supercritical fluid chromatography
    DOI:  https://doi.org/10.1016/j.aca.2026.345263
  3. Clin Chem Lab Med. 2026 Mar 10.
    An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of linezolid in human serum and plasma.
    Marie Kubicova, Neeraj Singh, Carina Schuster, Friederike Bauland, Andrea Geistanger, Michael Vogeser, Judith Taibon.
       OBJECTIVES: The quantification of linezolid in serum and plasma matrices is crucial for therapeutic drug monitoring and pharmacokinetic studies. A reference measurement procedure (RMP) was developed and validated to provide accurate, precise, and reliable measurements, fit for its intended use while ensuring suitability for clinical and laboratory settings.
    METHODS: Linezolid quantification was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS), with specific ion transitions monitored for the analyte and its stable isotope-labeled internal standard (ISTD). The RMP validation process encompassed key performance parameters including selectivity, matrix effects (MEs), linearity, precision, accuracy, and stability. MEs were assessed by comparing slopes and correlation coefficients (r) across neat solution, analyte-free serum, native serum, and various plasma matrices. Precision and accuracy were quantified through multiday experiments and by analyzing spiked samples across multiple concentrations. Measurement uncertainties were calculated by determining standard uncertainties as well as expanded uncertainties for the entire concentration range.
    RESULTS: The described method provides robust quantification of linezolid in human serum and plasma across a concentration range of 0.0800-48.0 μg/mL, demonstrating high specificity and no significant matrix effects. A comprehensive five-day validation study (n=60) demonstrated the method's strong performance, with intermediate precision coefficients of variation (CVs) ≤2.1 % and repeatability CVs≤2.0 %. At the lower limit of the measuring interval (LLMI), the method maintained reliability, achieving an intermediate precision CV of 3.7 % and a repeatability of 1.7 % (n=20). Mean relative biases ranged from -2.0 to 2.9 %. Measurement uncertainty (MU) analysis further supported these results, with standard MUs (k=1) determined to be 1.6-3.8 % for single measurements and 0.8-1.4 % for target value assignments (n=6). Specific assessment at the LLMI yielded an expanded uncertainty (k=2) of 7.6 and 2.8 % for single measurements and target value assignments (n=6), respectively. These results confirm the method's suitability for accurate and precise quantification across the entire measuring range.
    CONCLUSIONS: This LC-MS/MS-based candidate RMP accurately quantifies linezolid in human serum and plasma. Its robust performance makes it suitable for standardization of routine analytical assays and for analysis of individual patient samples, ensuring analytical reliability and traceability.
    Keywords:  SI units; isotope dilution-liquid chromatography-tandem mass spectrometry; linezolid; qNMR characterization; reference measurement procedure; traceability
    DOI:  https://doi.org/10.1515/cclm-2025-1366
  4. Vet World. 2026 Jan;19(1): 15-28
    First validated liquid chromatography-tandem mass spectrometry method for simultaneous quantification of propranolol and 4-hydroxypropranolol in pig plasma and dried blood spots and its application to a pharmacokinetic study.
    Anisa Bardhi, Domenico Ventrella, Alberto Elmi, Ronette Gehring, Davide Martelli, Ilaria Troisio, Maria Laura Bacci, Andrea Barbarossa.
       Background and Aim: Propranolol is a widely used non-selective beta-adrenergic blocker in human medicine, with well-characterized pharmacokinetics (PK) in humans but virtually no data available for pigs, a species of growing biomedical relevance. Furthermore, no validated bioanalytical methods exist for propranolol or its primary metabolite, 4-hydroxy-propranolol, in porcine matrices. This study aimed to develop and validate a rapid, sensitive, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of propranolol and 4-hydroxypropranolol in pig plasma and dried blood spots (DBS), and to apply it in a preliminary PK investigation in pigs.
    Materials and Methods: Sample preparation involved simple protein precipitation (plasma) or solvent extraction (DBS) using acetonitrile-water mixtures, followed by chromatographic separation on a Bridged ethyl hybrid C18 column (50 × 2.1 mm, 1.7 μm; 4-min run). Detection was performed in Multiple reaction monitoring mode with propranolol-d7 as the internal standard. Validation followed EMA ICH M10 guidelines, assessing linearity, accuracy, precision, matrix effects, recovery, and stability. The method was then applied to plasma samples from five juvenile female pigs receiving oral propranolol (3 mg/kg, q8 h).
    Results: The method demonstrated excellent linearity (r2 > 0.99) and acceptable accuracy and precision (±15%) across 2-500 ng/mL (propranolol) and 1-400 ng/mL (4-hydroxypropranolol). Recoveries ranged from 83% to 116% (plasma) and 81%-113% (DBS), with no matrix interference or carry-over. In vivo PK data revealed rapid absorption (Tmax 1.14 ± 0.63 h), moderate elimination (t½ 2.19 ± 0.86 h), and a mean Cmax of 112.02 ± 81.87 ng/mL. Notably, 4-hydroxypropranolol was undetectable in all plasma samples, suggesting species-specific metabolic differences.
    Conclusion: This study reports the first validated LC-MS/MS assay for propranolol and 4-hydroxypropranolol in pigs and demonstrates its successful application in a PK study. The method's simplicity, short runtime, and compatibility with DBS microsampling make it ideal for preclinical and veterinary research, minimizing animal stress and sampling volume. Absence of 4-hydroxypropranolol highlights interspecies metabolic variability and warrants further investigation into propranolol biotransformation pathways in swine and other translational models.
    Keywords:  4-hydroxypropranolol; beta-blocker; dried blood spots; liquid chromatography–tandem mass spectrometry; microsampling; pharmacokinetics; pigs; propranolol
    DOI:  https://doi.org/10.14202/vetworld.2026.15-28
  5. Metabolomics. 2026 Mar 07. pii: 38. [Epub ahead of print]22(2):
    Polar microcystins or arginine methyl ester can serve as sensitive reference materials for system suitability tests in untargeted metabolomics using reversed-phase LC-MS: A case study.
    Tommy Melzer, Georg Pohnert, Nico Ueberschaar.
       INTRODUCTION: LC-MS system suitability test (SST) is crucial for reliable data acquisition especially in untargeted metabolomics.
    OBJECTIVES: Identification of best reference materials (RMs) to improve best quality assurance (QA) and quality control (QC) practices.
    METHODS: Investigations were performed using a C18 reversed-phase (RP) column LC-MS approach.
    RESULTS: Targeted cyanotoxin analysis revealed a performance loss of the used C18 RP column although the SST confirmed a fit for purpose instrument which prompted to test several additional RMs.
    CONCLUSION: QA procedures for LC-MS can be improved by incorporating polar microcystins or arginine methyl ester as RMs for SST.
    Keywords:  LC-MS; Quality assurance; Reference materials; System suitability test; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/s11306-026-02413-9
  6. Se Pu. 2026 Mar;44(3): 349-356
    [Simultaneous determination of selinexor， posaconazole， venetoclax， and voriconazole in human plasma using ultra-high performance liquid chromatography-tandem mass spectrometry].
    Xiong Xiao, Yue Li, Jin-Fang Hu, Qing Wan, Hong-Wei Peng.
      Drugs used to treat hematologic malignancies often exhibit narrow therapeutic windows， significant inter-individual variability， and high risks of drug-drug interactions， necessitating therapeutic drug monitoring （TDM） for individualized dosing. For patients requiring concomitant antifungal therapy due to infections arising from severe chemotherapy-induced myelosuppression， TDM can optimize plasma concentrations of both antineoplastic and antifungal agents. This concurrent optimization ensures anti-tumor efficacy while effectively mitigating the risk of invasive fungal infections， positioning TDM as a critical component of personalized management in hematologic malignancies. This study established and validated a rapid ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） method for the simultaneous quantification of the antineoplastic agents selinexor （SEL） and venetoclax （VEN）， along with the antifungal agents voriconazole （VOR） and posaconazole （POSA） in human plasma. Chromatographic separation was achieved using a Kinetex® XB-C18 column （50 mm × 3.0 mm， 2.6 µm） with a mobile phase consisting of methanol and 10 mmol/L ammonium acetate containing 0.1% formic acid， delivered at a flow rate of 0.5 mL/min under gradient elution conditions. The injection volume was 2 μL， and the total run time was 4.0 min. Detection employed an electrospray ionization source operating in positive ion mode with multiple reaction monitoring （MRM）. Following optimization of mass spectrometric parameters， the method underwent comprehensive validation. Calibration curves demonstrated excellent linearity （r>0.994） over the ranges of 0.04-1.48 μg/mL for SEL， 0.15-5.50 μg/mL for VEN， 0.29-11.77 μg/mL for VOR， and 0.15-6.05 μg/mL for POSA. Intra-day and inter-day precisions （RSDs） were ≤7.1% at all concentration levels. Extraction recoveries were ≥85.3%， demonstrating efficient and consistent sample processing. Method accuracy， determined by the percentage deviation of measured concentrations from nominal values， fell within the acceptable range of 87.4% to 109.0% across all QC levels， confirming the method's trueness. The practical utility of the validated UHPLC-MS/MS method was demonstrated through its application to clinical specimens. We collected and analyzed 81 clinical samples from 30 patients with acute myeloid leukemia treated with the combination of SEL and VEN. Measured plasma concentrations of SEL in these patients ranged from 0.049 μg/mL to 0.646 μg/mL. Notably， significant inter-individual variability in the peak plasma concentration （Cmax） of SEL was observed within the same treatment cycle for approximately 30% of the patient cohort. This study offers evidence-based support for personalized precision therapy in this patient population， which exhibits substantial inter-individual variability and complex drug-drug interactions in clinical practice.
    Keywords:  selinexor; therapeutic drug monitoring; triazole antifungal agent; ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）; venetoclax
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.05003
  7. J Pharm Biomed Anal. 2026 Mar 09. pii: S0731-7085(26)00123-8. [Epub ahead of print]275 117455
    Detection and quantification of ezetimibe and its major glucuronide in patients with hepatic impairment via liquid chromatography-tandem mass spectrometry.
    Dominique O Farrera, Max M Maloney, Christopher S LaMadrid, Paige Lenzen-Hammerel, Lauren R Radtke, Nathan J Cherrington.
      Following administration of ezetimibe (EZE), levels of the glucuronidated metabolite ezetimibe-glucuronide (EZEG) are altered in patients with hepatic impairment. A rapid and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) method was developed and validated to quantify EZE and EZEG in human plasma and urine. Samples were prepared using a protein precipitation procedure with methanol containing stable isotope-labeled internal standards (EZE-d4 and EZEG-d4). Separation of the analytes was achieved using acetonitrile-water (0.1% formic acid) as the mobile phase at a flow rate of 0.5 mL/min on a C18 column. The analytes were detected using negative ionization in multiple reaction monitoring (MRM) mode. The mass transition pairs of m/z 408.4→271.0 and m/z 584.5→271.0 were used to quantify EZE and EZEG, respectively. The method was linear over a concentration range of 1.5 ng/mL - 1 µg/mL for EZE and EZEG in the plasma. In the urine, EZE was detectable a linear range of 5 ng/mL - 1 µg/mL and EZEG ranged from 3 ng/mL - 1 µg/mL. The method was validated in accordance with U.S. Food and Drug Administration and European Medicines Agency regulatory standards, including specificity, sensitivity, stability, repeatability and reproducibility. This ensures accuracy and reliability of test results which thereby enhances patient safety and helps support clinical decisions.
    Keywords:  Biomarker; Ezetimibe (EZE); Ezetimibe glucuronide (EZEG); Hepatic impairment; LC–MS/MS; Protein precipitation
    DOI:  https://doi.org/10.1016/j.jpba.2026.117455
  8. J AOAC Int. 2026 Mar 07. pii: qsag018. [Epub ahead of print]
    Quantitative Determination Method for Residual Colistin A/B in Edible Meats and Milk by Liquid Chromatography-Tandem Mass Spectrometry.
    Takayuki Nakajima, Chieko Nagano, Hiroki Okubo, Hiroshi Koike, Yumi Ohba.
       BACKGROUND: Colistin is a polypeptide antibiotic serving as a crucial "last-resort" agent against multidrug-resistant Gram-negative bacteria.
    OBJECTIVE: This study aimed to develop a reliable analytical method for the determination of colistin A and B residues in food matrices using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
    METHOD: Test portions were extracted with methanol-water (1:1) under hydrochloric acid-acidified conditions, followed by a secondary extraction with 0.1 mol/L hydrochloric acid. The extracts were cleaned using a weak cation-exchange cartridge, and chromatographic separation was performed on a biphenyl column with gradient elution, followed by LC-MS/MS.
    RESULT: The developed method produced sharp and symmetrical peaks for colistin A and B, with no significant matrix effects. Recoveries ranged from 81.0% to 105.9% across all analytes and matrices, and both repeatability and reproducibility met the target criteria specified in the Japanese guidelines for method validation. The limits of quantification were 20 ng/g in bovine and swine muscles for colistin A and 10 ng/g for others.
    CONCLUSION: An LC-MS/MS method was established for quantitative determination of colistin A and B residues in food matrices. The validation results met all performance criteria of the Japanese guidelines, confirming that the method provides reliable and precise measurements suitable for food residue monitoring.
    HIGHLIGHTS: Reliable quantification of colistin residues was achieved using three distinct retention modes, enabling reliable monitoring of residues in foods.
    DOI:  https://doi.org/10.1093/jaoacint/qsag018
  9. Se Pu. 2026 Mar;44(3): 234-247
    [Determination of 30 homologues of phosphatidylcholines and lysophosphatidylcholines in human serum by liquid chromatography-tandem mass spectrometry and their correlation analysis with coronary artery disease].
    Wen-Yu Li, Zhao-Yang Liu, Jun Dong, Rui-Yue Yang, Hong-Xia Li, Wen-Xiang Chen, Si-Ming Wang.
      Phosphatidylcholine （PC） and lysophosphatidylcholine （LPC） homologues are closely associated with coronary atherosclerosis. Accurate determination of their contents can provide an important basis for the clinical diagnosis and prognosis of coronary artery disease （CAD）. In this study， an analytical method based on liquid chromatography-tandem mass spectrometry was established， which enabled the simultaneous and accurate determination of 30 PC and LPC homologues using only 10 μL of human serum. Methanol-acetonitrile-methyl tert-butyl methyl ether-water was used as the extraction system， and an XBridge C18 column was selected as the stationary phase. The mobile phase consisted of an acetonitrile-water mixture （1∶1， volume ratio） and isopropanol， both containing 7.5 mmol/L ammonium formate and 0.15% （volume ratio） formic acid， and gradient elution was adopted for separation. Detection was performed using an electrospray ionization source in the positive ion mode with multiple reaction monitoring. Method validation results showed that the method exhibited a good linear relationship， with an average linear correlation coefficient of ≥0.999 7 over a linear range of 0.125-100 μg/mL. The limits of detection and limits of quantification were 0.01-1.94 μg/mL and 0.03-6.48 μg/mL， respectively. The recoveries ranged from 85.4% to 114.3%， while the intra-day precision and inter-day precision were no more than 4.6% and 12.6%， respectively. Serum samples from 110 clinical volunteers who underwent coronary angiography were determined using this method. The average population concentration of PC homologues was 526.80 μg/mL， and that of LPC homologues was 73.67 μg/mL. Spearman correlation analysis revealed that PC and LPC homologues were closely correlated with the severity of CAD， as well as with related clinical biochemical and lipid metabolism indicators， suggesting that they could serve as potential CAD-related metabolites in clinical practice. Designed to meet clinical analysis needs， this method features small serum sample volume， simple operation， and excellent response. It can efficiently determine 30 PC and LPC homologues in human serum， providing an important reference for exploring the association between these two lipid classes and CAD， as well as the translational application of related biomarkers.
    Keywords:  atherosclerosis; coronary artery disease; lipidomics; liquid chromatography-tandem mass spectrometry （LC-MS/MS）; lysophosphatidylcholine; phosphatidylcholine
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.08011
  10. mSystems. 2026 Mar 12. e0145925
    S'Wipe: user-friendly stool collection for high-throughput gut metabolomics and multi-omics.
    Dana Moradi, Ali Lotfi, Alexey V Melnik, Aleksandr Smirnov, Konstantin Pobozhev, Hannah Monahan, Evguenia Kopylova, Yanjiao Zhou, Alexander A Aksenov.
      The microbiome is increasingly recognized as a key factor in health. Intestinal microbiota modulates gut homeostasis via a range of diverse metabolites. In particular, molecules such as short-chain fatty acids (SCFAs), the microbial fermentation products of dietary fiber, have been established to be reflective of microbiome and/or dietary shifts, and SCFAs alterations have been linked to multiple gastrointestinal disorders, from cancer to colitis. Despite their potential as biomarkers, technical challenges in stool collection have limited clinical translation. Here, we present Stool Wipe (S'Wipe), an ultra-low-cost fecal collection method using lint-free, mass spectrometry (MS)-compatible cellulose wipes as toilet paper. Specimens are preserved in ethanol without refrigeration and can be shipped via regular mail. Mass spectrometry analysis demonstrated that S'Wipe captures both volatile and non-volatile metabolites with reproducibility and stability validated for diagnostically relevant molecules. We show that S'Wipe performs equivalently to direct stool collection, enabling interchangeable use and comparison with existing studies. This methodology is ideally suited for large-scale population studies, longitudinal tracking, and personalized medicine applications.
    IMPORTANCE: Gut microbiome and intestinal metabolome present invaluable diagnostic and therapeutic targets. However, conventional stool testing has several barriers, limiting bioassessment from populations. Routine, high-temporal-resolution monitoring of stool metabolome, including extensively validated and broadly informative biomarkers such as short chain fatty acids (SCFAs), is not implemented due to relatively high cost and inconvenience of sampling, possible need for clinical setting for sample collection, difficulty in collecting samples reproducibly-especially due to potential for user errors-requirement for freezer storage and maintenance of the cold chain during shipment. We present a sampling strategy specifically designed to overcome these obstacles. We demonstrate how this method can enable capturing accurate molecular snapshots at massive scales, at ultra-low cost. The approach collapses complex medical-grade collection into easy self-administration. Individuals can thereby self-monitor therapeutic responses through routine metabolome tracking, including the volatilome, otherwise hindered by infrastructure restrictions. Ultimately, this sampling approach is intended to enable participatory wellness transformation through practical high-frequency self-sampling.
    Keywords:  disease biomarkers; economical; fecal metabolome; gas chromatography-mass spectrometry; gastrointestinal diagnostics; health monitoring; liquid chromatography-mass spectrometry; low cost; metabotyping; patient self-testing; personalized medicine; personalized nutrition; sample collection; short-chain fatty acids
    DOI:  https://doi.org/10.1128/msystems.01459-25
  11. Biomed Chromatogr. 2026 Apr;40(4): e70400
    Development and Validation of a UPLC-MS/MS Method for the Determination of Apalutamide in Human Plasma and Its Application in a Bioequivalence Study.
    Qi Wen, Juan Cai, Min Yang, Xin-Ying Xu, Bo-Ping Wei, Qiang Wu, Dai-Guo Zhao.
      A UPLC-MS/MS method was developed for determining apalutamide (APT) in human plasma, applied to a bioequivalence study of two tablet formulations. Plasma samples were pretreated by protein precipitation with acetonitrile using APT-13C-d3 as an internal standard. Separation was on a Waters BEH C18 column with gradient elution (0.1% formic acid in water/acetonitrile) at a flow rate of 0.50 mL/min. ESI (+) and MRM transitions were employed, with m/z 478.1 → 221.2 for APT and m/z 482.2 → 225.3 for APT-13C-d3. A crossover bioequivalence study (single-dose, two-period) was conducted under fasting or fed conditions. A good linear relationship was observed over the concentration range of 6.00~1200.00 ng/mL. The intrabatch and interbatch accuracy ranged from 95.06% to 104.22% (RSD ≤ 5.05%). The recovery was not less than 102.36%, and no significant matrix effect was observed for APT and APT-13C-d3. In both fed and fasting trials, the 90% CI for the geometric mean ratios of Cmax and AUC0-72 between the test and reference formulations fell within the range of 80.00%~125.00%. This method is simple, rapid, and accurate, making it suitable for the determination of APT concentration in human plasma. Additionally, the test and reference APT tablets were demonstrated to be bioequivalent.
    Keywords:  UPLC‐MS/MS; apalutamide; bioequivalence; human plasma; pharmacokinetics
    DOI:  https://doi.org/10.1002/bmc.70400
  12. Anal Bioanal Chem. 2026 Mar 10.
    Simple and sensitive cellulose paper-based microextraction of 25 steroid esters from human serum/plasma for doping control purposes.
    Monica Mazzarino, Wim Van Gansbeke, Aðalheiður Dóra Albertsdóttir, Pieter Van Renterghem, Peter Van Eenoo.
      Blood is increasingly used as a biological matrix in the doping control field; however, the limited volume typically available for analysis poses a significant challenge, particularly when multiple tests must be carried out on the same sample. This study proposes a simple and efficient microextraction protocol based on the use of unmodified cellulose for the extraction of 25 steroid esters from just 20 µL of serum or plasma. Sample preparation consisted of spotting 20 µL of serum or plasma on a cellulose card, followed by a double extraction of the target analytes with 500 µL of methanol and subsequent derivatization using Girard's Reagent P. The analysis was carried out using liquid chromatography coupled with tandem mass spectrometry. The analytical workflow was qualitatively validated according to the UNI CEI EN ISO/IEC 17025 standard and the WADA technical rules and notes in terms of selectivity (target analytes were distinguishable from the matrix interferences), sensitivity (limits of detection in the range of 0.05-0.70 ng/mL), carry-over (no signals were detected in the negative sample injected after the positive sample), intra and inter-day stability of the retention times (≤0.5%), matrix effect (13-32%), extract stability (the target analytes were stable for at least 72 h in the autosampler at 10 °C), and extraction yield (43-88%). Plasma samples collected after testosterone undecanoate injection were finally analyzed: the compound was detectable in all three volunteers evaluated for at least 1 month after administration, demonstrating the effectiveness of the newly developed method for doping control purposes.
    Keywords:  Cellulose card; Doping control analysis; Microextraction techniques; Plasma; Serum; Steroid esters
    DOI:  https://doi.org/10.1007/s00216-026-06426-0
  13. Clin Chem Lab Med. 2026 Mar 12.
    An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of 17-hydroxyprogesterone in human serum and plasma.
    Martina Bachmann, Myriam Ott, Neeraj Singh, Friederike Bauland, Daniel Köppl, Alexander Gaudl, Andrea Geistanger, Uta Ceglarek, Manfred Rauh, Judith Taibon.
       OBJECTIVES: Accurate measurement of 17α-hydroxyprogesterone (17-OHP) in human serum and plasma is required for steroid profiling, to ensure the appropriate diagnosis of congenital adrenal hyperplasia. Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) to quantify 17-OHP in human serum and plasma has been developed.
    METHODS: A two-dimensional heart-cut LC approach was used to minimize matrix effects and prevent co-elution of interferences. A post-column infusion experiment was conducted to assess the potential impact of different matrices on ion suppression or enhancement effects. A multi-day validation experiment assessed precision, trueness and accuracy. Measurement uncertainty was evaluated in compliance with current guidelines.
    RESULTS: The working range of this RMP was 0.0200-60.0 ng/mL (0.0605-182 nmol/L) and the method proved to be matrix-independent. The intermediate precision coefficient of variation (CV) ranged from 1.5 % to 2.1 % and repeatability CV from 1.3 % to 1.9 %, across all concentration levels. The relative mean bias ranged from -4.0 % to 2.0 %, regardless of the matrix and concentration level. The measurement process for single measurements showed an expanded uncertainty (k=2) between 3.6 % and 4.5 %. This uncertainty was further reduced performing the target value assignment (n=6), resulting in a range of 2.0-2.5 %. Equivalence between the candidate RMP and the Joint Committee on Traceability in Laboratory Medicine-listed RMPs was established through participation in the External Quality Assessment scheme for reference Laboratories in Laboratory Medicine (RELA) ring trial.
    CONCLUSIONS: The performance of this procedure enables the evaluation and standardization of routine assays, and the evaluation of patient samples, to ensure traceability of individual patient results.
    Keywords:  17α-hydroxyprogesterone; SI units; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability
    DOI:  https://doi.org/10.1515/cclm-2025-1169
  14. Se Pu. 2026 Mar;44(3): 267-275
    [Performance validation and consistency assessment of three liquid chromatography-tandem mass spectrometry methods for detecting fat-soluble vitamins].
    Bing-Chu Li, Bing-Hui Li, Zhi-Yang Chen, Zi-Yang Li, Mei-Ling Yin, Xing Lyu, Zhong-Yuan Xiang, Qi-Chen Long, Min Hu.
      Accurate quantification of fat-soluble vitamin A （VA）， vitamin D （25（OH）D）， vitamin E （VE）， vitamin K （VK） is essential for health assessment and disease diagnosis. Liquid chromatography-tandem mass spectrometry （LC-MS/MS） plays a crucial role in the measurement of these vitamins due to its high specificity and sensitivity. However， the comparability and concordance of the detection results among different LC-MS/MS methods are still suboptimal. In this study， the analytical performance including linearity， limit of detection， limit of quantitation， precision， accuracy and carryover was evaluated according to CLSI C62 and the Suggestions on Clinical Application of Liquid Chromatography-Mass Spectrometry. Aliquots of 40 patient samples were collected from August to October 2022 from the Second Xiangya Hospital， Central South University. These fat-soluble vitamins were measured using three LC-MS/MS methods. Passing-Bablok regression， Bland-Altman plots and concordance correlation coefficient （CCC） were performed to compare the differences among three methods. The consistency of the results before and after recalibration with a unified calibrator was evaluated. The results showed that the total CVs for the three mass spectrometry kits were 2.7%-10.1%， and the recoveries for all kits ranged from 88.8% to 109.2%， indicating that their detection performance met the requirements for clinical application. Concordance correlation analysis showed that two of the three methods had substantial agreement in vitamin D testing （CCC： 0.938）， while the CCCs for other methods ranged from 0.322 to 0.853. After recalibration with a unified calibrator， except for vitamin K， the CCCs for the results of the three methods ranged from 0.918 to 0.983. In conclusion， the performance validation results of three LC-MS/MS methods for fat-soluble vitamins meet clinical requirements. The consistency among the three methods was poor， but using a unified calibrator significantly improved the consistency of these methods. The study provides recommendations for the standardization of clinical fat-soluble vitamin testing， thereby contributing to improved accuracy and consistency of results. This study will provide more reliable evidence for clinical diagnosis and treatment.
    Keywords:  calibration; consistency; fat-soluble vitamins; liquid chromatography-tandem mass spectrometry （LC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.04002
  15. Se Pu. 2026 Mar;44(3): 248-256
    [Establishment and international comparison of reference methods for glycated hemoglobin].
    Mo Wang, Shun-Li Zhang, Rui Zhang, Yi-Chuan Song, Jie Shi, Qiao-Zhen Xue, Yan-Wei Hu.
      To promote the standardization and harmonization of glycated hemoglobin A1c （HbA1c） testing results in medical laboratories across Beijing， we established a reference method for HbA1c. This method participated in the International Comparison Program for HbA1c Reference Laboratories organized by the European Reference Laboratory Network for HbA1c to validate its accuracy. The experimental samples consisted of HbA1c network reference laboratory proficiency testing samples imported annually from the Netherlands， including six calibrators （A-F）， ten intercomparison samples， several quality control materials， and additional auxiliary samples with International Federation of Clinical Chemistry and Laboratory Medicine （IFCC）-assigned values. The pre-treatment process involved digesting samples with protein endopeptidase Glu-C. The experimental samples were removed from the -80 ℃ freezer and allowed to reach room temperature. Subsequently， 50 μL of Glu-C （mass concentration： 200 μg/mL） was added to each sample vial at a ratio of total hemoglobin to enzyme of 1 mg∶0.01 mg. The volume was then adjusted to a final volume of 500 μL with ammonium acetate solution （50 mmol/L， pH 4.3）. After thorough mixing， the samples were incubated at 37 ℃ for 18-20 h. Using mobile phases of methanol and 0.1% formic acid aqueous solution under gradient elution conditions， the detection of HbA1c in international samples was performed via high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）. A gradient elution mode was employed for liquid chromatography separation using a Shimadzu C18 column （50 mm×3 mm， 2.2 μm）. The injector temperature was maintained at 4-8 ℃， with a column temperature of 30 ℃. The flow rate was kept constant at 0.6 mL/min， with an injection volume of 5 μL and a total run time of 8 min. The MS detection was performed using electrospray ionization （ESI） in positive ion mode with multiple reaction monitoring （MRM）. The monitored ion pairs for the precursor and product ions of non-glycated and glycated N-terminal hexapeptides are m/z 348.2/237.2 and m/z 429.2/245.2， respectively. The linearity was evaluated by performing regression analysis. The HbA1c （mmol/mol） quantification in unknown samples was achieved by performing linear regression using Analyst 1.6.2 software. Peak areas were integrated， with the abscissa （X-axis） representing the known concentration ratio of HbA1c to hemoglobin A0 （HbA0）， and the ordinate （Y-axis） representing the average peak area ratio of glycated to non-glycated hexapeptides. Both glycated and non-glycated hexapeptides eluted within 3 min， with linear correlation coefficients ranging from 0.999 6 to 0.999 8. The quality control （QC） materials （≥3 types） annually provided by the IFCC were analyzed. Since each QC material in every shipment typically consists of two replicated vials， enzymatic digestion was performed in two separate batches. For each batch， five parallel samples were processed， followed by triplicate injections of each sample. Intra-assay and total coefficients of variation （CV） were calculated. The intra-assay coefficients of variation （CVs） were 0.35%-2.20%， and the total CVs were 0.83%-2.39%. From 2018 to 2024， IFCC compared individual laboratory results with the overall median， calculating proportional bias （slope） and systematic bias （intercept） based on linear regression. Our laboratory's combined statistical test value （computed from slope and intercept） ranged from 0.1 to 3.0. The systematic bias ranged from -0.69 to 1.27， and the proportional bias ranged from -0.009 to 0.021. The residual values from all testing points in the 2024 proficiency testing （PT） program were evenly distributed around zero. Furthermore， the magnitude of residual deviations from zero was relatively small compared to those observed across the 15 participating laboratories， demonstrating satisfactory consistency in our laboratory's analytical results. The established HbA1c reference method demonstrated robust performance， achieving satisfactory results in IFCC international comparisons from 2018 to 2024.
    Keywords:  glycated hemoglobin; international comparison; liquid chromatography-tandem mass spectrometry （LC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.03017
  16. Clin Chim Acta. 2026 Mar 07. pii: S0009-8981(26)00144-0. [Epub ahead of print]587 120962
    Performance evaluation of automated magnetic beads extraction method for the measurement of 23 plasma steroids using dual liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
    Han Chen, Gang Xu, Mei-Juan Zhang, Fu-Rong Ying, Hai-Xia Huang, He-Huan Liu, Jian-Rong Yang.
       OBJECTIVES: To evaluate the performance of magnetic beads extraction method (MGE) for quantifying plasma steroids using liquid chromatography tandem mass spectrometry (LC-MS/MS).
    METHODS: The evaluation encompassed calibration linearity, limit of detection (LOD), lower limit of the measuring interval (LLMI), specificity and matrix effects, trueness and recovery, intra- and inter-day precisions, repeatability, dilution consistency and stability of 23 plasma steroids extracted by MGE method were evaluated. The 23 plasma steroids were isolated and analyzed through a single sample preparation and two injections by LC-MS/MS.
    RESULTS: All 23 steroids were successfully resolved chromatographically within 12.1 min. The automated MGE method exhibited excellent linearity (R2 > 0.995 for all analytes) with coefficients of variation (CVs) ranging from 3.36% to 9.28% at LLMI and from 1.58% to 16.53% at LOD. Additionally, the CVs and average deviation for repeatability were between 0.55% and 7.11%, and - 6.57% to 10.81%, respectively. Both intra-day precision (0.88% to 8.43%) and inter-day precision (0.98% to 6.51%) satisfied the acceptance criteria. The average deviation for trueness and recovery ranged from -13.93% to 9.06%. Moreover, specificity, matrix effect, dilution consistency, and stability were distinctly identified and conformed to guideline requirements. Notably, levels of AD, T, E1, and 17-OHP were significantly elevated in patients with polycystic ovary syndrome (PCOS) (p < 0.05).
    CONCLUSION: The automated MGE method demonstrates high efficiency and reliability for the simultaneous quantification of 23 plasma steroids, offering a promising solution for high-throughput analysis of steroid panels in clinical in the future.
    Keywords:  Automation; LC-MS/MS; MGE; Steroids
    DOI:  https://doi.org/10.1016/j.cca.2026.120962
  17. Se Pu. 2026 Mar;44(3): 312-328
    [Establishment and application of a knowledge-directed pseudo-targeted analytical method for diabetic retinopathy].
    Yan-Qing Hou, Xiang-Yu Chi, Ting-Hu DU, Zeng-Qi Yan, Dai-di Hou, Chun-Xiu Hu, Yue-Xing Liu, Xin-Yu Liu, Guo-Wang Xu.
      Diabetic retinopathy （DR） is a common blinding eye disease caused by diabetes mellitus and is the leading cause of acquired vision loss in adults worldwide. DR is asymptomatic in its early stages， and patients often miss the optimal treatment window by the time they seek medical attention due to vision impairment. Traditional methods used for DR diagnosis have inherent limitations and are not conducive to large-scale rapid screening. Biomarkers can reflect the stage of the disease owing to their specificity and sensitivity， which is crucial for the early diagnosis of DR. In the present study， 142 potential literature-based biomarkers associated with DR were included in a knowledge-directed strategy. And metabolites with different physicochemical properties were chromatographically separated using a 100-mm Discovery HS F5 column. A pseudo-targeted metabolomics method based on ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） that simultaneously scans positive and negative ions was established to improve analysis coverage and throughput. The method was validated using eight representative isotope-labeled internal standards as analytical targets. All isotope-labeled internal standards exhibited satisfactory linearities in both positive- and negative-ionization modes， with linear dynamic ranges spanning over three orders of magnitude and correlation coefficients （r²） above 0.995. Extraction recoveries ranged between 75% and 108% at three distinct concentration levels with relative standard deviations （RSDs） below 13%. Notably， 91% of the isotope-labeled internal standards exhibited intra-day precision with RSDs of less than 5% across both ionization modes. Similarly， 91% of the analytes demonstrated inter-day precision with RSDs of less than 10%， with all below 16.3%， indicating good method precision. The developed method was used to analyze 137 serum samples， including 40 DR-free patients with diabetes mellitus （NDR） and 97 patients with DR to investigate the practicality of the method. Quality control （QC） samples were used to evaluate the data， which revealed that the instrument was stable during the analytical sequence. Partial least squares discriminant analysis （PLS-DA） models were constructed to identify and differentiate between the metabolic profiles of the DR and NDR groups with the aim of providing a statistical basis for the classification and diagnosis of DR based on metabolic differences observed in the serum samples. The NDR group and DR groups of varying clinical grades were well separated. A total of 85 differential metabolites were identified between NDR and DR groups using nonparametric tests. Further analysis led to the selection of choline and 12-hydroxyeicosatetraenoic acid （12-HETE） as two markers that effectively distinguished the DR and NDR groups. In addition， the markers exhibited a good ability to distinguish between DR and NDR patients when used in combination. The pseudo-targeted metabolomics-based knowledge-directed method developed in this study provides a reference for screening and diagnosing DR.
    Keywords:  diabetic retinopathy （DR）; disease markers; metabolomics; ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.02011
  18. J Forensic Sci. 2026 Mar 08.
    Differentiation of cannabinoid isomers via Cu-mediated molecular ion formation and electrospray ionization-tandem mass spectrometry.
    Alleigh N Couch, Christopher M Zall, J Tyler Davidson.
      Gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) remains the primary analytical technique used for cannabis analysis in seized drug laboratories. Electron ionization (EI) mass spectra exhibit extensive fragmentation, enabling the identification of cannabinoids by comparison with reference EI mass spectral libraries. However, limitations such as thermal degradation and potential cannabinoid conversion can occur due to the elevated temperatures of the GC inlet. In contrast, liquid chromatography-mass spectrometry (LC-MS) uses a soft ionization technique, such as electrospray ionization (ESI), which predominantly yields the protonated molecule with minimal fragmentation. Even with collisional activation using tandem mass spectrometry (MS/MS) analysis, the product ion spectra are nearly identical for cannabinoid isomers, reducing the effectiveness of this technique for cannabinoid identification. In this study, copper (Cu) salts are used to induce cannabinoid molecular ion formation under ESI conditions, enabling cannabinoid isomer differentiation. Thirteen cannabinoids were analyzed in the presence of Cu, and the resulting MS/MS product ion spectra exhibited fragmentation analogous to cannabinoid EI mass spectra. To evaluate forensic applicability, the EI-like product ion spectra were searched against the NIST 20 EI-MS mass spectral library using NIST MS Search software. Spectral matches confirmed that this alternative approach can generate EI-like data under ESI-MS/MS conditions, improving cannabinoid isomer identification. Additionally, this method was applied to methanolic extracts of authentic cannabis plant material to ensure cannabinoid molecular ion formation in real-world samples. The developed method offers an alternative approach to traditional workflows, while providing spectral data consistent with those routinely interpreted by seized drug analysts.
    Keywords:  NIST MS search; cannabinoids; electrospray ionization‐tandem mass spectrometry (ESI‐MS/MS); fragmentation; isomer differentiation; molecular ion formation
    DOI:  https://doi.org/10.1111/1556-4029.70290
  19. Anal Chim Acta. 2026 May 01. pii: S0003-2670(26)00210-2. [Epub ahead of print]1397 345260
    Preparation of styrene-maleic anhydride copolymer nanobrush chromatographic stationary phase and its application in phospholipid analysis of exosomes.
    Ping Wang, Meijuan Xue, Xinhui Guan, Xiaofei Gu, Xiaoqiang Qiao.
       BACKGROUND: Phospholipids in serum exosomes are promising biomarkers for breast cancer diagnosis. However, the separation of phospholipids by liquid chromatography presents several challenges due to their inherent structural heterogeneity and the presence of isomeric forms. Traditional columns often struggle with the overlapping retention times of phospholipid species, which can result in poor resolution and difficulty in accurate quantification. Furthermore, the varying polarities and hydrophobicities of different phospholipid classes necessitate specialized stationary phases to achieve optimal separation. This study aimed to develop a novel chromatographic material for the comprehensive analysis of phospholipids in complex biological matrices.
    RESULTS: In this study, a novel silica-based Sil-SMA-GMS copolymer brush stationary phase was fabricated for the first time via reversible addition-fragmentation chain transfer (RAFT) polymerization, using styrene-maleic anhydride (SMA) copolymer as the functional matrix and glycerol monostearate (GMS) as the derivatization reagent. Benefiting from the synergistic effects of hydrophobic moieties (benzene rings and long alkyl chains) and hydrophilic groups (carboxyl and hydroxyl groups) in the SMA copolymer, the prepared stationary phase exhibited a unique mixed hydrophilic interaction chromatography/reversed-phase liquid chromatography (HILIC/RPLC) retention mechanism. It demonstrated excellent separation selectivity and stability for both hydrophilic and hydrophobic small molecules, achieving a maximum column efficiency of 81,029 N/m. Notably, this stationary phase overcame the limitations of traditional single-mode chromatographic columns, enabling the simultaneous separation and analysis of phospholipids that vary in class, acyl chain length, and degree of unsaturation. Furthermore, by coupling the Sil-SMA-GMS column with mass spectrometry (MS), a non-targeted analytical method for serum exosomal phospholipids was developed. Applying this method to serum exosomes from breast cancer patients and healthy controls led to the identification of 9 major classes encompassing 200 phospholipid molecular subtypes, as well as the screening of phospholipid markers closely associated with breast cancer pathogenesis.
    SIGNIFICANCE: This work presents a novel chromatographic material for the efficient separation of phospholipids in complex biological samples and establishes a technical foundation for the construction of multidimensional lipidomics platform. This advancement provides crucial support for the screening of cancer-related biomarkers, elucidating tumor metabolic mechanisms, and exploring targeted therapeutic targets.
    Keywords:  Breast cancer biomarkers; Copolymer brush stationary phase; Mixed-mode chromatography; RAFT polymerization; Serum exosome phospholipids
    DOI:  https://doi.org/10.1016/j.aca.2026.345260
  20. Talanta. 2026 Mar 09. pii: S0039-9140(26)00304-8. [Epub ahead of print]305 129648
    Validated LC-MS/MS method for the quantification of glyphosate and its metabolites in human serum: A biomonitoring study in farming intensive regions of the Western Cape, South Africa.
    Luthando L Tiya, Rianita van Onselen, Annalise Zemlin, Eric Decloedt, Tracy Kellermann.
      Glyphosate, a widely used herbicide approved in over 100 countries for its broad-spectrum efficacy and low cost, has raised increasing environmental and health concerns. Monitoring inadvertent exposure requires quantification of glyphosate and its metabolites, aminomethylphosphonic acid (AMPA) and methylphosphonic acid (MPA). The Western Cape Province of South Africa, characterized by a Mediterranean climate that supports intensive agriculture, lacks biomonitoring data on these compounds. This study reports the first validated LC-MS/MS method in Southern Africa for the simultaneous quantification of glyphosate, AMPA, and MPA in human serum without derivatization, and its application to clinical samples from farming-intensive areas of the Western Cape. Calibration standards ranged from 15.0 to 760 ng/mL for glyphosate, 25.0-1600 ng/mL for AMPA, and 0.75-48 ng/mL for MPA, using Oasis MAX cartridges for extraction. Analysis was performed on a Sciex QTRAP 6500+ LC-MS/MS system with a Phenomenex Luna C8 column and gradient elution. Method validation followed SANTE/2020/12830, REV.2 guidelines, assessing linearity, limit of quantification, selectivity, matrix effects, recovery, precision, and stability. Among 258 serum samples opportunistically collected from clinics located in regions with intensive pesticide use, glyphosate and/or its metabolites were detected in 11.6% of samples, with three exhibiting quantifiable concentrations. This robust, non-derivatized LC-MS/MS method enables accurate biomonitoring of glyphosate exposure and provides the first evidence of human exposure in agricultural communities of the Western Cape.
    Keywords:  Aminomethylphosphonic acid; Glyphosate; Health risk; LC-MS/MS; Metabolites; Pesticides
    DOI:  https://doi.org/10.1016/j.talanta.2026.129648
  21. J Pharmacol Toxicol Methods. 2026 Mar 05. pii: S1056-8719(26)00012-2. [Epub ahead of print] 108419
    Simultaneous quantification of venetoclax, busulfan and voriconazole in plasma by LC-MS/MS: Clinical applications in patients with acute myeloid leukemia.
    Zhenhua Zhang, Bingchen Ge, Yanyu Wang, Changrui Long, Huixing Wu, Hui Chen, Shijian Xiang, Benjie Zhou.
       OBJECTIVE: To develop a novel approach for quantitative analysis of three pharmaceutical agents commonly employed in the treatment of hematological disorders (venetoclax, busulfan, and voriconazole) by liquid chromatography tandem mass spectrometry (LC-MS/MS).
    METHODS: The internal standards were venetoclax-d8 (venetoclax), busulfan-d8 (busulfan), and posaconazole (voriconazole), which were separated on a Phenomenex Kinetex® C18 (2.1 × 50 mm, 2.6 μm) column, and the multiple reaction monitoring was carried out in the positive ion mode of the SCIEX Triple Quad™ 5500. Protein precipitation pretreatment was performed using acetonitrile, with solvent A (2 mmol/L ammonium acetate dissolved in water containing 0.1% formic acid) and solvent B (acetonitrile solution containing 0.1% formic acid) as mobile phases. The flow rate was 0.8 mL/min, and the injection volume was 5 μL.
    RESULTS: The method was validated with regards to selectivity, lower limit of quantification, linearity, precision and accuracy, matrix effect and extraction recovery, dilution integrity, and stability. The runtime of each plasma sample was 3.1 min. The concentration ranges of the calibration curves were 50-10,000 ng/mL for venetoclax and voriconazole, and 15-3000 ng/mL for busulfan, with all the three correlation coefficients greater than 0.999. There were significant drug-drug interactions with venetocalx and voriconazole, and the incidence of adverse reactions was related to the venetoclax exposure in vivo. Concentrations of busulfan and voriconazole showed substantial interindividual variability.
    CONCLUSION: This study develops, with validation, a novel LC-MS/MS method for simultaneous determination of venetoclax, busulfan, and voriconazole, which is suitable for application to therapeutic drug monitoring in patients with acute myeloid leukemia.
    Keywords:  Busulfan; LC-MS/MS; Therapeutic drug monitoring; Venetoclax; Voriconazole
    DOI:  https://doi.org/10.1016/j.vascn.2026.108419
  22. J Chromatogr A. 2026 Mar 07. pii: S0021-9673(26)00215-3. [Epub ahead of print]1774 466885
    Shared-autosampler parallel LC-MS/MS platform for high-throughput multi-analyte quantification in complex matrices.
    Shanshan Cai, Qisheng Zhong, Jiaqi Liu, Lisong Chen, Lingling Shen, Hongyuan Hao, Ting Zhou.
      Modern analytical tasks increasingly require the simultaneous quantification of large numbers of chemically diverse analytes in complex matrices, which remains challenging for a single LC-MS method. Here, we developed a shared-autosampler parallel LC-MS/MS strategy that enables two fully independent chromatographic methods to be executed sequentially within a single analytical cycle while generating one unified chromatogram-mass spectrum. The system employed two LC pump modules and two columns to form two independent LC pathways, which shared one autosampler and one MS detector for selective MRM acquisition and subsequent signal integration. Integration was performed exclusively at the MS detection and data-processing level, without inter-dimensional coupling or analyte transfer between columns. This avoided the mobile-phase compatibility and dead-volume limitations associated with 2D-LC, enabled modular recombination of chromatographic and MS conditions, and improved analytical throughput without additional MS detector or autosampler hardware. The performance of the strategy was demonstrated in two applications. An acidic/basic dual-method configuration enabled quantitative analysis of 394 emerging contaminants from 14 chemical classes in human serum within 28 min, exhibiting good linearity, limits of quantification not exceeding 0.20 ng/mL for >90 % of analytes, and peak-area RSDs generally below 10 %. A HILIC/RP parallel-column combination achieved simultaneous determination of 27 hypoglycemic agents spanning a log P range of -6.8 to 5.9 in a single 20 min run, reducing analysis time by >50 % compared with standard methods, with good precision and recoveries in spiked food samples. Overall, the shared-autosampler parallel LC-MS/MS strategy provided a flexible and efficient platform for high-throughput multi-analyte quantification in complex matrices.
    Keywords:  Complex matrices; High-throughput analysis; Multi-analyte quantification; Parallel chromatography; Shared-autosampler LC–MS/MS
    DOI:  https://doi.org/10.1016/j.chroma.2026.466885
  23. Molecules. 2026 Feb 27. pii: 799. [Epub ahead of print]31(5):
    Rapid Analysis of Phytic Acid by Paper Spray Mass Spectrometry.
    Ping Guo, Sijie Zhu, Bo Chen.
      Phytic acid (PA), owing to its strong acidity and multidentate metal-chelating properties, readily forms multiple adduct/complex ions in mass spectrometry and is prone to pronounced matrix effects, resulting in complicated spectra and compromised sensitivity and quantitative robustness, which poses a major challenge for rapid and accurate PA quantification. Herein, we developed a rapid quantitative method for PA based on trimethylsilyldiazomethane (TMSD) methyl-ester derivatization coupled with paper spray mass spectrometry (PS-MS). PA was derivatized with TMSD to yield the methylated product (PA-Me), and the derivative solution was purified via "post-derivatization nitrogen blow-down followed by water reconstitution", thereby markedly reducing background interference. In positive-ion mode, the stable sodium adduct ion [PA-Me+Na]+ (m/z 851.04) was used as the quantifier, enabling fast quantification with selected ion monitoring (SIM). PS-MS was performed with a 15 μL spotting volume and methanol/water (90/10, v/v, containing 0.1% formic acid) as the spray solvent, allowing rapid analysis without chromatographic separation. The method exhibited good linearity over 0.125-30 μg/mL (R2 ≥ 0.9965), with a limit of detection (LOD, S/N = 3) of 0.080 μg/mL and a limit of quantification (LOQ, S/N = 10) of 0.270 μg/mL. The intra-day and inter-day precision values were both < 10% (RSD), and recoveries ranged from 87.2% to 122.4%. This LC-free strategy features low solvent consumption and high analytical throughput, and was validated using rice bran protein and rice bran polysaccharide samples, providing technical support for rapid screening and quality control of PA in complex food/plant matrices.
    Keywords:  derivatization; high-throughput analysis; paper spray mass spectrometry; phytic acid; rice bran products
    DOI:  https://doi.org/10.3390/molecules31050799
  24. J Chromatogr A. 2026 Feb 28. pii: S0021-9673(26)00181-0. [Epub ahead of print]1774 466851
    Chiral derivatization reagents for enantioselective LC-MS/MS analysis of amino acids with analyte-specific fragmentation.
    Tatsuya Sakamoto, Kana Ishikura, Mayu Onozato, Takeshi Fukushima.
      Accurate enantioselective analysis of amino acids by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains challenging, as existing derivatization strategies often compromise either chromatographic resolution or structural discrimination in tandem mass spectrometry. In this study, we developed novel chiral derivatization reagents, (R)- and (S)-Oqic, based on a 1,3-oxazinoquinoline-4-one scaffold, for the simultaneous determination of amino acid enantiomers via LC-MS/MS. The reagents reacted rapidly with amino acids under mild basic conditions to form their chiral derivatives, enabling the simultaneous separation of 37 amino acids (18 pairs of amino acid enantiomers and Gly) on a conventional ODS column within 16 min. The resulting derivatives exhibited unique fragmentation patterns, yielding characteristic fragment ions at m/z 190 and [M + H - 171], with intensity ratios dependent on the tagged amino acid, thereby facilitating reliable analyte discrimination. The method was validated and subsequently applied to the analysis of commercially available fermented dairy products, revealing the presence of d-Ala, d-Ser, d-Asp, d-Glu, and d-Arg in yogurt and a probiotic beverage. The detection of sweet-tasting d-amino acids (d-Ala, d-Ser, and d-Arg) suggests their potential contribution to the sensory properties of fermented foods. Overall, the developed Oqic-based method provides a powerful tool for elucidating bacterial metabolism and food flavor profiles.
    Keywords:  Chiral derivatization; LC–MS/MS; dl-Amino acid
    DOI:  https://doi.org/10.1016/j.chroma.2026.466851
  25. Se Pu. 2026 Mar;44(3): 357-362
    [Rapid determination of venetoclax in plasma by ultra performance liquid chromatography-tandem mass spectrometry].
    Ying Zhu, Xiao-Li Ma, Song-Lin Yu, Ling Qiu.
      Leukemia is a malignant tumor of the hematological system characterized by the uncontrolled proliferation of abnormal hematopoietic cells in the bone marrow. It often presents with anemia， bleeding tendency， infection risk and organ invasion. These clinical symptoms bring severe survival risks to patients. Although traditional chemotherapy regimens are effective in the treatment of some hematological malignancies， their efficacy is limited for elderly patients， those with high-risk genetic characteristics or comorbidities. In recent years， targeted drugs have revolutionized the treatment of leukemia. By selectively inducing tumor cell apoptosis， they have significantly improved the remission rate and survival prognosis of patients with multiple leukemia subtypes. Venetoclax is a B-cell lymphoma 2 （BCL-2） inhibitor and plays an important role in the clinical treatment of hematological malignancies， such as acute myeloid leukemia and chronic lymphocytic leukemia. In addition， it also shows potential efficacy in other hematological malignancies such as multiple myeloma and mantle cell lymphoma. Although the efficacy of venetoclax is remarkable， the individual differences in blood drug concentration are significant due to factors such as drug interactions， polymorphisms of metabolic enzymes， and liver and kidney function. Venetoclax exhibits significant inter-individual pharmacokinetic differences， the trough concentration is significantly correlated with the treatment response， and if the peak concentration exceeds the warning concentration， adverse reactions will be triggered. Clinical trials have reported a variety of adverse events associated with venetoclax， including neutropenia， tumor lysis syndrome， thrombocytopenia， infection， anemia， diarrhea， nausea， upper respiratory tract infection， cough and musculoskeletal pain. Therefore， to minimize the risk of adverse events in the clinical use of venetoclax as much as possible， it is necessary to reasonably guide its clinical dosage. Therapeutic drug monitoring can optimize individual dosing regimens by measuring the steady-state concentration in patients' blood. This research aims to establish a rapid， sensitive and reliable ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method. This method is used for the quantitative determination of venetoclax in plasma， and its performance was validated. This method employs an electrospray ionization and multiple reaction monitoring （MRM） in the positive ion mode to detect venetoclax and its isotope internal standard venetoclax-d8. Methanol was used for protein precipitation， C18 reversed-phase chromatographic column was used for liquid phase separation. Gradient elution was performed using acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The flow rate was 0.4 mL/min， the run time was 3.5 min， and the retention time of venetoclax was 1.95 min. The analysis time is short， facilitationg the rapid determination of clinical samples. Dosage escalation is commonly adopted in the treatment of venetoclax. In this study， a pretreatment approach involving extraction followed by dilution was used. This methodincreased the upper limit of quantification and expanded the linear range. The linear range of venetoclax was 50-10 000 ng/mL， r2>0.999. The method had good specificity. The intra-run precision and inter-run precision were 1.8%-4.5% and 2.7%-6.1%， respectively， the recoveries were 100.3%-102.9%， the matrix effects ranged from 88.0% to 111.0%， and the carryover was less than 20% of the minimum concentration of linear range. This method was applied to the therapeutic drug monitoring of venetoclax in acute myeloid leukemia （AML） patients， and the peak and trough concentrations of venetoclax were obtained for different patients to monitor their blood drug concentration. The above research results indicate that this method can accurately and robustly quantify venetoclax in human plasma， which helps address the drug monitoring needs of leukemia patients receiving venetoclax treatment and guide clinical personalizd therapy.
    Keywords:  plasma; therapeutic drug monitoring; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）; venetoclax
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.06002
  26. Metabolomics. 2026 Mar 07. pii: 35. [Epub ahead of print]22(2):
    Multiplexed LC-MS analysis reveals novel insights into grapevine defense mechanisms by expanding metabolome coverage.
    Pedro G Vásquez-Ocmín, Amelie Pérez, Ana Romeo-Oliván, Virginie Puech-Pages, Sylvie Fournier, Bernard Dumas, Alban Jacques, Guillaume Marti.
       INTRODUCTION: Grapevine trunk diseases (GTDs), such as esca, pose a major threat to viticulture worldwide and are associated with complex biochemical responses in woody tissues. Comprehensive metabolome coverage remains a challenge, as conventional methods often overlook non-polar metabolites critical to plant defense mechanisms.
    OBJECTIVES: This study aimed to expand metabolome and lipidome coverage of grapevine wood by integrating complementary LC-MS approaches, in order to identify metabolic signatures linked to pathogenic fungi and to a biocontrol agent.
    METHODS: Woody tissues of Vitis vinifera cv. Cabernet-Sauvignon were inoculated with Phaeomoniella chlamydospora, Phaeoacremonium minimum, and/or the biocontrol fungus Trichoderma atroviride (Vintec®). A biphasic extraction was coupled with three orthogonal LC-MS methods-reverse-phase (RP), hydrophilic interaction chromatography (HILIC), and lipidomics-focused RP. Data were processed through the MSCleanR workflow and integrated using the DIABLO multi-block statistical framework. Compound classification was performed with NPClassifier.
    RESULTS: The multiplexed strategy enabled the annotation of 1,425 unique features, representing an 83% increase compared to previous studies. Distinct metabolomic and lipidomic signatures were associated with fungal infection and biocontrol treatments. Lipidomic analysis highlighted oxidized fatty acids (oxylipins) -specifically hydroxy-eicosatetraenoic acids (13-HETE, 16(R)-HETE, and 11(R)-HETE)-as potential signaling molecules in defense responses. NPClassifier revealed diverse biosynthetic classes, including phenylpropanoids, terpenoids, and sphingolipids, underscoring the chemical heterogeneity of grapevine responses.
    CONCLUSION: This multiplexed LC-MS workflow provides a versatile analytical pipeline for untargeted metabolomics and lipidomics in plants. By integrating complementary methods, the study uncovered novel biomarkers of grapevine defense, particularly oxylipins, emphasizing the critical role of lipidomics in deciphering plant-pathogen interactions.
    Keywords:  Esca; Lipidomics; Metabolomics; Oxylipins
    DOI:  https://doi.org/10.1007/s11306-026-02410-y
  27. Mass Spectrom (Tokyo). 2026 ;15(1): A0191
    Enantioselective Determination of Carnitine Enantiomers in Food and Supplement Samples by Chiral Liquid Chromatography-Tandem Mass Spectrometry.
    Aogu Furusho, Yutaro Nakayama, Hiroki Mizuno, Eiji Sugiyama, Kenji Kojima, Kenichiro Todoroki.
      Carnitine (CAR) is an essential compound for animals and plays several physiological roles related to energy production. These functions are possessed by only the l-forms, while the d-forms are known to inhibit the uptake of the l-forms, thus causing a CAR deficiency. Therefore, it is required to determine dl-CAR in various samples such as foods, supplements, and drugs by an enantioselective analytical method. In this study, we developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using derivatization. The CAR enantiomers derivatized with 4-nitrophenylhydrazine were successfully separated on a CHIRALPAK ZWIX (-) column (resolution = 1.64) and detected with high sensitivity and selectivity by MS/MS. The developed method was well validated and applied to the analysis of eight kinds of foods and an l-CAR supplement. Only the l-forms were found in all the tested food samples and were abundant in meat, milk, and yogurt. The system was also applicable for a supplement sample and could detect the d-CAR contamination at the 2.5% level, suggesting its potential for use in the purity test of CAR preparations.
    Keywords:  chiral separation; derivatization; food analysis
    DOI:  https://doi.org/10.5702/massspectrometry.A0191
  28. Expert Rev Proteomics. 2026 Mar 14.
    Towards large-scale mass spectrometry-based omics for clinical applications.
    Maor Arad, Leonard J Foster.
       INTRODUCTION: As healthcare advances toward personalized medicine, mass spectrometry-based research is advancing our understanding of cellular biology and disease states, and translating these findings into clinical applications. This review highlights recent advances in methodology and technology that demonstrate the capabilities of mass spectrometry-based proteomics, lipidomics, and metabolomics in clinical practice.
    AREAS COVERED: The ability to directly analyze functional molecules with mass spectrometry uncovers crucial clinical information. Each data modality (proteins, lipids, and metabolites) provides essential insight into healthy and disease states. As technology advances, integrating data from different modalities unlocks new possibilities for clinical research. To gain the most from this multi-omic data, unsupervised integration methods can provide detailed insights into complex biological processes. As the field applies this knowledge, healthcare could experience significant leaps in the near future. This review examines recent advancements in mass spectrometry-based proteomics, lipidomics, and metabolomics, focusing on how improvements in sample preparation, automation, and multi-omics data integration are making large-scale clinical studies more accessible.
    EXPERT OPINION: Recent technical and methodological advancements in mass spectrometry analysis have propelled healthcare toward a tipping point, shifting from traditional RNA- and DNA-based research to downstream analysis of protein, lipid, and metabolite effectors.
    Keywords:  Automation; lipidomics; liquid chromatography; mass spectrometry; metabolomics; multi-omics; personalized medicine; proteomics; robotics; unsupervised data integration
    DOI:  https://doi.org/10.1080/14789450.2026.2646657
  29. J Sep Sci. 2026 Mar;49(3): e70385
    Ultra-Selective Ultra-High-Performance Liquid Chromatography-Triple-Stage Mass Spectrometry Assay for Quantification of Pyraclostrobin in Zebrafish Liver Tissues Via Multistage Fragmentation to Enhance Sensitivity.
    Chunpeng Feng, Yufeng Guo, Yunlong Sheng, Meichen Liu, Jiale Liu, Yue Deng, Shuang Feng, Yalin Xi, Meiyun Shi, Lei Yin.
      Pyraclostrobin, a widely applied methoxyacrylate fungicide, poses considerable risks to aquatic ecosystems due to its environmental persistence and bioaccumulation potential. Understanding its tissue-specific residue profile in aquatic organisms such as zebrafish is essential for elucidating ecotoxicological mechanisms and evaluating environmental safety. In this study, a novel, rapid, and sensitive ultra‑high‑performance liquid chromatography-triple‑stage mass spectrometry (UHPLC‑MS3) method was developed and fully validated for the quantification of pyraclostrobin in zebrafish liver tissue. The assay utilized an MS3 transition (m/z 388.0 → 194.0 → 164.0), which significantly enhanced selectivity and sensitivity. The method achieved fast separation (3 min per sample) and exhibited excellent linearity from 1 to 300 ng/mL (R2 ≥ 0.998). Precision (intra‑ and inter‑day coefficient of variation <10.2%), recovery (94.4%-95.4%), and matrix effects (99.2%-108.9%) all met rigorous bioanalytical criteria. This work represents the first application of UHPLC‑MS3 for strobilurin analysis in aquatic tissues, providing a reliable high‑throughput platform for residue monitoring and mechanistic toxicology research.
    Keywords:  UHPLC‐MS3; liver tissues; pyraclostrobin; zebrafish
    DOI:  https://doi.org/10.1002/jssc.70385
  30. Anal Chem. 2026 Mar 09.
    Resolving sn-Positional Isomers of Docosahexaenoic Acid-Bound Phospholipids in the Mouse Brain by Cyclic Ion Mobility Mass Spectrometry Imaging.
    Seiya Tanaka, Emi Sasaki, Taiji Kawase, Thanai Paxton, Kenji Hirose, Aya Yoshinaga-Kiriake, Kazuaki Yoshinaga, Yugo Iwasaki, Naohiro Gotoh, Katsuyoshi Masuda.
      Phospholipids containing docosahexaenoic acid (DHA, 22:6) are crucial for brain function and are abundant in the brain. While 22:6 predominantly associates with the sn-2 position of phosphatidylcholine (PC) in the plasma and liver, the brain contains a characteristic PC in which 22:6 is bound to the sn-1 position. However, the precise relationship between the 22:6-binding site and its distribution in the brain remains unclear. Ion mobility mass spectrometry imaging can visualize phospholipid molecular species in the brain. However, the distribution of structurally similar phospholipids, such as PC sn-positional isomers, has not been fully elucidated. Here, we used cyclic ion mobility coupled mass spectrometry imaging to distinguish PC isomers, specifically PC(22:6/16:0) and PC(16:0/22:6), after 30 passes of cyclic ion mobility, and revealed their differential distribution in the mouse brain using a stable isotope-labeled 22:6. We observed significant differences between the brain distribution of the isomers in mice orally administered 22:6-incorporated PCs. The reliability of the results was further supported by quantitative data obtained using liquid chromatography-tandem mass spectrometry. This study demonstrates for the first time that cyclic ion mobility mass spectrometry imaging can effectively distinguish and visualize structurally similar isomers, such as phospholipid sn-positional isomers with different distributions.
    DOI:  https://doi.org/10.1021/acs.analchem.5c06621
  31. Se Pu. 2026 Mar;44(3): 257-266
    [Development of a liquid chromatography-tandem mass spectrometry method for steroid hormone detection during pregnancy and consistency analysis with chemiluminescence immunoassay].
    Wei-Xiang Wu, Li-Hong Wu, Fu-Qiang Diao, You-Wen Luo, Chun-Ming Gu, Ming-Yong Luo.
      Steroid hormones play a critical role in maintaining pregnancy and supporting fetal development. Accurate quantification of these hormones is essential for evaluating the endocrine status during pregnancy. While chemiluminescence immunoassay （CLIA） is widely used in clinical practice in China， it has inherent methodological limitations. In contrast， liquid chromatography-tandem mass spectrometry （LC-MS/MS） has been internationally recognized for its superior analytical performance but remains underutilized in Chinese obstetric practice. Moreover， systematic evaluations comparing these two methods in pregnant women are still lacking. This study aimed to develop and validate an isotope-dilution LC-MS/MS method for the simultaneous quantification of four key steroid hormones， including cortisol， dehydroepiandrosterone sulfate （DHEAS）， testosterone， and 17-hydroxyprogesterone （17-OHP）. In addition， a consistency analysis was conducted between LC-MS/MS and CLIA among pregnant women. Method validation followed international bioanalytical guidelines， evaluating linearity， limits of quantification （LOQs）， recovery， and precision. Serum samples from 94 pregnant women were collected at Guangdong Women and Children Hospital between January 2022 and December 2023， and were analyzed using both LC-MS/MS and CLIA. Method comparison was performed using paired t-tests， Pearson correlation， Bland-Altman plots， and Passing-Bablok regression to assess concentration differences， correlation， and systematic biases between the two methods. In this study， the established LC-MS/MS method exhibited excellent performance， with linear coefficients of determination >0.999， LOQs of 0.008-0.137 ng/mL， spiked recoveries of 92.1%-110.9%， and intra-assay relative standard deviations （RSDs） of 3.2%-9.0%， meeting the requirements for clinical detection. In a cohort of 94 pregnant women， paired t-tests revealed significant differences in hormone concentrations measured by LC-MS/MS and CLIA （all P<0.001）. Specifically， LC-MS/MS yielded significantly lower values for cortisol （64.31 vs. 120.60 ng/mL） and DHEAS （532.44 vs. 1 612.1 ng/mL）， but higher values for testosterone （1.10 vs. 0.88 ng/mL） and 17-OHP （2.00 vs. 1.07 ng/mL）. Bland-Altman analysis further revealed obvious negative proportional biases for cortisol and DHEAS， with bias values of -56.29 ng/mL and -1 079.68 ng/mL， respectively. Additionally， the biases increased with the increase of concentration. In contrast， testosterone and 17-OHP showed positive biases， with bias values of 0.22 ng/mL and 0.89 ng/mL， respectively. The two methods showed extremely strong positive correlations in the detection of the four hormones （P<0.001， r=0.819-0.974）， among which the correlation of testosterone detection results was the highest （r=0.974）. In the Passing-Bablok regression analysis， the consistency regression equation for cortisol was CLC-MS/MS=-3.58+0.57CCLIA. The confidence interval （CI） of the slope did not include 1， indicating obvious proportional bias. The consistency regression equation for DHEAS was CLC-MS/MS=-59.77+0.38CCLIA. The CIs of the intercept and slope did not include 0 and 1， respectively， suggesting the existence of fixed and proportional biases. The consistency regression equation for testosterone was CLC-MS/MS=-0.05+1.30CCLIA. The CIs of the intercept and slope did not include 0 and 1， respectively， indicating the existence of fixed and proportional biases. In addition， the differences increased with the increase of concentration. For 17-OHP， the regression equation was CLC-MS/MS=0.10+1.75CCLIA. Though the intercept was not statistically significant， the slope deviated significantly from 1， indicating a proportional bias. Additionally， LC-MS/MS values were significantly higher， and the difference increased with rising concentration. In conclusion， the established LC-MS/MS method offers high sensitivity， precision， and accuracy for the simultaneous quantification of multiple steroid hormones. The method reliably measured cortisol， DHEAS， testosterone， and 17-OHP levels in maternal serum. Additionally， our results revealed statistically significant differences between the two methods across all four hormones in pregnant women， with biases varying by concentration. These findings highlight the importance for clinicians to consider methodological differences when interpreting hormone test results during pregnancy， in order to improve the accuracy of endocrine assessment and reduce the risk of misdiagnosis due to analytical discrepancies. Future studies should incorporate large-scale maternal cohorts covering all stages of pregnancy and high-risk populations to further assess the clinical applicability of different methods and promote the standardization of LC-MS/MS for obstetric endocrine monitoring. In parallel， trimester-specific reference intervals should be established to support clinical interpretation and risk evaluation.
    Keywords:  chemiluminescence immunoassay; consistency analysis; liquid chromatography-tandem mass spectrometry （LC-MS/MS）; steroid hormone; testosterone
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.07001
  32. Int J Mol Sci. 2026 Feb 25. pii: 2128. [Epub ahead of print]27(5):
    Changes in Metabolism and Lipid Composition with Nitrogen Starvation and Recovery in a New Productive Strain of Neochlorella semenenkoi Using N15-Isotopic Labeling and HRMS.
    Anna Vishnevskaya, Anton Bashilov, Dmitry Senko, Sergey Osipenko, Maria Sinetova, Nikita Malyshev, Philipp Khaitovich, Eugene Nikolaev, Yury Kostyukevich.
      Microscopic green algae are active producers of beneficial compounds, particularly those containing nitrogen. However, the metabolism of nitrogen-containing compounds is diverse and depends on the conditions of the nitrogen source. As a result, the approach to studying the metabolism of nitrogen-containing compounds becomes more complicated. This work demonstrates the metabolic changes in the high-productive green algae Neochlorella semenenkoi IPPAS C-1210 under conditions of nitrogen starvation and subsequent reintake, using high-performance liquid chromatography-mass spectrometry (HPLC-MS) with 15N isotopic labeling. The presented results include semi-quantitative chromatography-mass spectrometric analysis for 17 amino acids, a metabolomic profile of over 40 isotopically labeled compounds, an assessment of metabolic flux via isotopic incorporation, and an analysis of cellular lipid composition under varying growth conditions. The findings indicate that this strain can utilize ammonium acetate as a nitrogen source, consuming nitrogen in the ammonium form. The degree of isotopic labeling in compounds often diverged significantly from their quantitative changes (concentrations and chromatographic peak areas), suggesting that isotopic analysis may offer advantages over purely quantitative analysis for biological systems. Furthermore, in vivo biological isotopic labeling is shown to assist in identifying compounds absent from standard mass spectrometric databases.
    Keywords:  HPLC-MS; lipidomics; metabolomics; microalgae; nitrogen starvation; stable isotope labeling
    DOI:  https://doi.org/10.3390/ijms27052128
  33. Anal Chim Acta. 2026 May 08. pii: S0003-2670(26)00175-3. [Epub ahead of print]1398 345225
    Resolving lipid isomers up to the double bond position level using reversed-phase chromatography, EAD fragmentation, and high-resolution mass spectrometry: Application to pancreatic cancer.
    Marlene Pühringer, Leonida M Lamp, Denisa Kolářová, Milan Vošmik, Lisa Panzenboeck, Gunda Köllensperger, Michal Holčapek, Jürgen Hartler, Evelyn Rampler.
       BACKGROUND: Electron-activated dissociation (EAD) is a radical-based fragmentation technique that provides detailed structural information on lipids in a single spectrum, including double-bond positions, sn-1/sn-2 assignment, and molecular species. Its broader application, however, remains limited by the low intensity of diagnostic fragments. An alternative computational approach, called LC=CL, which is an extension of the software Lipid Data Analyzer (LDA), leverages retention time (RT) information obtained by reversed-phase liquid chromatography (RPLC) to identify ω-positions in intact lipid species.
    RESULTS: Here, we present an integrated RPLC-EAD-TOF-MS/MS workflow that combines LC=CL's RT-based with EAD's fragmentation-based annotation for deep structural characterization using a ZenoTOF 7600. This strategy was validated using both unlabeled and uniformly 13C-labeled yeast extracts. By combining the benefits of retention-time identification and radical-induced fragmentation, our method enables the reliable identification of double-bond locations and sn-positional isomers across 15 lipid classes.
    SIGNIFICANCE: In the analysis of plasma samples from pancreatic ductal adenocarcinoma (PDAC) patients and healthy controls, we demonstrate the power of our strategy for unambiguously resolving lipid isomers, revealing structure-specific patterns of dysregulated lipids. A total of 353 lipids were identified across 15 classes composed of glycerolipids, glycerophospholipids, and sphingolipids. Our findings not only confirmed known biomarkers, but also revealed additional chain isomers (e.g., SM 18:1; O2/20:0 and SM 16:1; O2/22:0), where only one of them was differentially regulated. Moreover, we discovered novel double-bond location-specific regulations, such as differential regulation of PC 16:0/18:3(n-6) in PDAC patients, whereas the isomer PC 16:0/18:3(n-3) did not exhibit any significant changes. Such an observation would have remained concealed using conventional methods. Accordingly, the presented RPLC-EAD-TOF-MS/MS platform facilitates detailed structural lipidomics in biologically and clinically relevant samples.
    Keywords:  Chromatography; Double bond localization; EAD; Fragmentation; LC=CL; Lipidomics; Mass spectrometry; Pancreatic cancer; RPLC-MS; Structural lipidomics
    DOI:  https://doi.org/10.1016/j.aca.2026.345225
  34. Anal Chim Acta. 2026 May 01. pii: S0003-2670(26)00235-7. [Epub ahead of print]1397 345285
    Faster, simpler, and more precise calibration curves: Expanding the scope of continuous calibration.
    Peter J H Williams, Nadini Thushara, Amin Yousefi, Harrison Mundschutz, Dennis K Hore, J Scott McIndoe.
       BACKGROUND: Accurate calibration curves are essential for quantitative analysis but are often neglected due to their time, cost, and labour demands. Traditional calibration involves measuring solutions of known concentrations to construct a calibration curve relating analyte concentration to instrument response. While these relationships rarely follow simple closed-form equations, linear approximations are commonly used for simplicity, often being fitted with too few data points for high-accuracy calibration. Continuous calibration addresses these challenges by continuously infusing concentrated calibrant into a clean matrix solution while monitoring the response online. This approach significantly reduces time and labour while generating extensive data, improving calibration precision and accuracy. Despite its advantages, method limitations and technical complexities have hindered widespread adoption.
    RESULTS: Here, continuous calibration is expanded and simplified with modern accessible equipment, open-source code, and a user-friendly web tool that together streamline experiments and data processing. The software generates smoothed and equation-fitted calibration curves complete with statistical-validity, quality-of-fit, and dynamic-range estimates. The method was demonstrated over a broad range of systems and analytical techniques, including external, standard addition, and internal standardization calibrations, and mass spectrometry, and infrared and ultraviolet-visible spectroscopies. The technique was also demonstrated to be more efficient and precise than discrete calibration. In ultraviolet-visible applications, molar absorption coefficients (ε) were obtained from a single experiment, with values for multiple analytes matching literature. For example, KMnO4/H2O analysis yielded ε = 2450 ± 40 M-1 cm-1 compared to the reported 2250-3340 M-1 cm-1. The approach was further extended to calibrate instrument response across pH ranges, yielding the literature pKa of a weak acid from a single experiment.
    SIGNIFICANCE: These advancements substantially improve the precision, efficiency, and accessibility of calibration, with the potential to enhance experimental quality and efficiency across many fields. We hope that these improvements encourage scientists to conduct high-quality calibration more frequently.
    DOI:  https://doi.org/10.1016/j.aca.2026.345285
  35. Se Pu. 2026 Mar;44(3): 276-285
    [Application of accuracy profile-based evaluation in the development of quantitative methods for urinary phenol detection].
    Xiao-Ning Liu, Yuan-Heng Shen, Wei-Feng Tang, Jun-Jie Ao, Jun-Xia Liu, Li Zhao, Li-Li Yuan, Xian Huang, Qian-Long Zhang.
      Accurate and reliable analytical methods are fundamental to ensuring the validity and reproducibility of scientific results， particularly in research involving trace-level contaminants in biological samples. While standardized methods regulated by official guidelines are commonly used in certified laboratories， research laboratories often rely on in-house or self-developed non-standard methods tailored for specific studies. These methods， however， frequently lack harmonized validation criteria， especially regarding accuracy evaluation. Traditionally， method accuracy is assessed through single performance indicators such as recovery， relative standard deviation （RSD）， or bias. These indicators， although useful， may vary widely in their interpretation and offer limited insight into overall method performance， particularly when comparing between laboratories or over different concentration ranges. To address these limitations， the Société Française des Sciences et Techniques Pharmaceutiques （SFSTP） introduced the accuracy profile approach. This method integrates both trueness and precision using a statistical model to generate β-expectation tolerance intervals， allowing a visual and comprehensive representation of whether an analytical method meets predefined acceptance criteria across multiple concentration levels. Unlike traditional evaluations， which may obscure marginal performance failures， the accuracy profile provides a single， unified framework that simplifies interpretation and decision-making. In this study， we applied the SFSTP accuracy profile approach to validate a high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） method for quantifying 20 urinary phenols. Method performance was assessed at three concentration levels （low， medium， and high） and the results were compared with conventional validation methods. The intra-batch RSD values ranged from 2.8% to 10.7%， and inter-batch RSD values ranged from 3.3% to 14.4%. The pooled intermediate precision RSD ranged from 4.9% to 16.6%， indicating acceptable reproducibility. Relative errors were between -25.3% and 13.0%， According to traditional evaluation using single parameters， the accuracy of all 20 phenol measurements met standard criteria. However， accuracy profile analysis revealed that only 13 phenols， such as bisphenol AF and bisphenol B， had β-expectation tolerance intervals entirely within the ±30% acceptance limits across all tested concentration levels. The remaining seven phenols， including benzylparaben and benzophenone-8， exceeded acceptable limits at low or medium concentrations， suggesting areas for method optimization. In conclusion， this study demonstrates that the HPLC-MS/MS method provides reliable quantitative results for the majority of tested urinary phenols. More importantly， the SFSTP accuracy profile offers a superior alternative to conventional validation methods by combining accuracy parameters and risk analysis into a coherent， statistically sound framework. This approach enables researchers to clearly identify the strengths and weaknesses of analytical methods without increasing experimental complexity or cost. Its advantages in standardization， interpretability， and decision-making support its broader adoption for validating non-standard methods in research and applied laboratories.
    Keywords:  accuracy assessment; high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）; urinary phenols; β-expectation tolerance intervals
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.04031
  36. Sci Rep. 2026 Mar 07.
    Bioanalysis of amphetamines in alternative matrices using a sensitive and validated liquid chromatography-tandem mass spectrometry method and its application to real samples.
    Humera Shafi Makhdoom, Ali Imran Abid, Nadeem Ul Hassan Khan, Zeerak Abbas.
      
    Keywords:  Amphetamines; Biological matrices; Toxicology; UHPLC-ESI-MS/MS; Validation
    DOI:  https://doi.org/10.1038/s41598-025-30861-1
  37. Se Pu. 2026 Mar;44(3): 226-233
    [Application of chromatography-mass spectrometry in the clinical analysis of multiple sclerosis].
    Zhen-Yu Yao, Yu-Fei Wang, Xiao-Wen Zhu, Hong Yang, Jia Wu, Guo-Jun Zhang.
      Multiple sclerosis （MuS） is a chronic， inflammatory， demyelinating disease of the central nervous system， with a globally increasing prevalence. Its pathogenesis involves complex interactions among immune dysregulation， genetic predisposition， and environmental factors. These contribute to a wide spectrum of clinical manifestations， posing significant challenges for both diagnosis and treatment. Metabolomics， the comprehensive analysis of small-molecule metabolites， has emerged as a powerful approach to elucidate MuS pathophysiology and identify potential biomarkers. In particular， alterations in amino acid and lipid metabolism are closely associated with inflammation， myelin damage， neurotransmitter imbalance， and immune activation. This review summarizes recent advancements in the application of chromatography-mass spectrometry techniques to MuS biomarker research. Key technologies include liquid chromatography-tandem mass spectrometry （LC-MS/MS） for analyzing metabolites in biological fluids， high resolution mass spectrometry （HRMS） for precise metabolite identification， gas chromatography-mass spectrometry （GC-MS） for profiling volatile compounds， and matrix-assisted laser desorption ionization-time of flight mass spectrometry （MALDI-TOF MS） for mapping spatial metabolite distributions in tissues. Metabolomic analyses of MuS patient samples have revealed significant disruptions in amino acid metabolism. For instance， reduced arginine levels and elevated asymmetric dimethylarginine levels in cerebrospinal fluid suggest vascular dysfunction. Altered tryptophan metabolism， including altered levels of kynurenic acid and 5-hydroxytryptophan， reflects neurotransmitter imbalances. Increased pyroglutamic acid levels indicate oxidative stress. Lipid metabolism is also markedly perturbed in MuS. Changes in palmitic acid levels affect cell membrane integrity， while alterations in sphingolipids， key components of myelin， are linked to demyelination. Elevated oxysterols correlate with neuroinflammation and immune dysregulation. Additionally， systemic lipid changes， such as reduced low-density lipoprotein and altered high-density lipoprotein cholesterol levels， have been associated with disease activity and progression. Due to their high sensitivity， resolution， and throughput， chromatography-mass spectrometry techniques are well-suited for detecting these metabolic alterations. Representative studies demonstrate the utility of LC-MS/MS in detecting amino acid perturbations in plasma and cerebrospinal fluid （CSF）， GC-MS in identifying lipid abnormalities in serum， HRMS in generating detailed brain tissue metabolite profiles， and MALDI-TOF MS in visualizing lesion-specific metabolic distributions. These findings have led to the identification of promising biomarker candidates. For instance， decreased arginine may indicate endothelial dysfunction， while elevated oxysterols may serve as markers of neuroinflammatory activity. Such biomarkers hold potential for facilitating early diagnosis， disease monitoring， and personalized treatment strategies. Metabolomics via chromatography-mass spectrometry technologies has significantly advanced our understanding of MuS pathophysiology. Amino acid dysregulation affects neurotransmitter synthesis and immune modulation， while lipid imbalances impair myelin integrity and cellular signaling. These insights provide a foundation for the development of metabolite-based diagnostic tools and therapeutic interventions. Nonetheless， several challenges remain. MuS exhibits high heterogeneity， and metabolomic data are complex and variable. Improved biomarker specificity and standardization are needed for clinical application. Technological advances in chromatography-mass spectrometry resolution， along with standardized protocols， will be essential. Large-scale clinical validation across MuS subtypes is also critical. Integrating metabolomics with genomics and environmental factors may enhance biomarker reliability and elucidate individual disease trajectories. In conclusion， this review highlights the crucial role of chromatography-mass spectrometry-based metabolomics in MuS research. By unraveling the metabolic underpinnings of inflammation and demyelination， it provides valuable insights into disease mechanisms and introduces novel avenues for early diagnosis and targeted therapy. Collaborative research efforts and methodological rigor will be key to translating these discoveries into clinical impact.
    Keywords:  amino acid metabolism; biomarkers; chromatography-mass spectrometry; lipid metabolism; multiple sclerosis （MuS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.03024
  38. Zhongguo Zhong Yao Za Zhi. 2025 Dec;50(24): 7013-7021
    [Pharmacokinetics of three pairs isomers of Caraway in Beagle dogs based on UPLC-MS/MS].
    Shu-Ping Li, Wahe-Fu Atikanmu, Abdu-la Rahima.
      This study established a rapid, sensitive, and convenient ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS) method for the simultaneous determination of three pairs of isomers(psoralen and isopsoralen, bavachin and isobavachalcone, bavachromene and isobavachromene) in Beagle dog plasma after oral administration of Caraway extract, and investigated their pharmacokinetic characteristics using a non-compartmental analysis method. Separation was achieved on a Phenomenex ACE Excel 3 phenyl column(4.6 mm × 75 mm, 3 μm) with a mobile phase of 0.2% formic acid in methanol and 0.2% formic acid aqueous solution under gradient elution. The three pairs of isomers and internal standards were completely separated within 15 min, and detection was performed in positive ion multiple reaction monitoring mode. The method was validated in terms of selectivity, linearity, precision, accuracy, recovery, matrix effects, and stability, meeting the requirements for quantitative analysis of biological samples. RESULTS:: showed that, after a single oral dose of Caraway in Beagle dogs, the T_(max) of psoralen, isopsoralen, isobavachalcone, bavachromene, and isobavachromene was 0.50-2.00 h, with a T_(1/2) of 1.55-7.08 h. Isopsoralen and psoralen exhibited relatively higher exposure, with C_(max) of 370.65 and 144.25 ng·mL~(-1), and AUC_(0-∞) of 646.17 and 273.34 ng·h·mL~(-1), respectively. Isobavachalcone, isobavachromene, and bavachromene showed relatively lower exposure, with C_(max) of 8.31, 1.61, and 0.74 ng·mL~(-1), and AUC_(0-∞) of 38.34, 5.48, and 2.24 ng·h·mL~(-1), respectively. Bavachin exposure was the lowest, and its pharmacokinetic parameters could not be determined. This study provides a scientific basis for the clinical application and subsequent new drug research of Caraway.
    Keywords:  Caraway; UPLC-MS/MS; isomers; pharmacokinetics
    DOI:  https://doi.org/10.19540/j.cnki.cjcmm.20250918.201
  39. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Feb 26. pii: S1570-0232(26)00085-1. [Epub ahead of print]1275 124996
    Hydrophobic covalent organic framework as a solid-phase microextraction coating for high-throughput quantification of endocannabinoids in Parkinson's disease rat plasma.
    Di Zhao, Chenqing Zhang, Zhiyu Zhang, Zhihong Chen, Yanmei Shi, Kai Hu.
      This study successfully synthesized a hydrophobic covalent organic framework (COF) material and applied it as solid-phase microextraction (SPME) coating for the detection of endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) in rat plasma. Under optimized extraction conditions, a high-throughput sample pretreatment method was established by integrating the prepared hydrophobic COF-SPME fibers with a 96-well plate platform, coupled with liquid chromatography-tandem mass spectrometry for analysis. The method enables the extraction of 96 samples within 25 min, significantly improving sample processing efficiency. In methodological validation, AEA and 2-AG showed good linearity in the ranges of 0.025-4.0 ng mL-1 and 0.1-10.0 ng mL-1, respectively, with correlation coefficients R2 ≥ 0.9988. The limits of detection for both analytes were in the range of 0.008-0.020 ng mL-1, recoveries ranged from 103.93% to 117.97%, and relative standard deviations were below 10.97%. When applied to plasma samples from Parkinson's Disease (PD) model rats, the results indicated significant alterations in AEA and 2-AG levels compared to the control group. This study demonstrates that the established method is suitable for rapid and highly sensitive detection of endocannabinoids in complex biological samples, providing a valuable analytical tool and reference for investigating the pathological mechanisms of PD.
    Keywords:  2-Arachidonoylglycerol; Endocannabinoid; LC-MS/MS; N-Arachidonoylethanolamine; Solid-phase microextraction
    DOI:  https://doi.org/10.1016/j.jchromb.2026.124996
  40. Se Pu. 2026 Mar;44(3): 296-301
    [Determination of vitamin A and vitamin E in human tear by liquid chromatography-tandem mass spectrometry and analysis of their relationship with dry eye].
    Bao-Bin Luo, Jing-Ran Jiao, Jing Bai, Xiang-Yi Liu.
      Dry eye is characterized as a multifactorial disorder affecting the tear film and ocular surface， leading to symptoms of discomfort and visual disturbances， with the potential for ocular surface damage. It is among the most prevalent ocular surface disorders， with an estimated prevalence ranging from 5.0% to 50.0%， and an overall prevalence between 21.0% and 50.4% in China. Despite its high global prevalence， the fundamental mechanisms underlying the pathology of dry eye remain largely unexplored. Vitamins are crucial for maintaining ocular surface homeostasis， with vitamins A and E being physiologically present and essential for cell differentiation， development， and proper function. Deficiencies in these vitamins are frequently associated with dry eye， and systemic supplementation has been shown to benefit patients with low vitamin intake. Vitamins A and E may have potential clinical predictive value for the diagnosis and treatment of dry eye. In this study， a liquid chromatography-tandem mass spectrometry （LC-MS/MS） method was developed to simultaneously measure the concentrations of vitamins A and E in human tears. A Phenomenex Kinetex C18 column was employed for gradient elution at a flow rate of 0.4 mL/min. The mobile phase A consisted of a 0.1% formic acid aqueous solution， while mobile phase B comprised a 2 mmol/L ammonium acetate and 0.1% formic acid methanol solution. Positive atmospheric pressure chemical ionization in multiple reaction monitoring mode was utilized for MS/MS detection. The analytical method for vitamin A demonstrated a validated linear range of 5.00-300.00 ng/mL， with a limit of detection at 2.00 ng/mL. The spiked recoveries were between 98.3% and 104.7%， and intra- and inter-day precision ranged from 1.2% to 7.0%. Similarly， the method for vitamin E exhibited a validated linear range of 25.00-1 000.00 ng/mL， with a limit of detection at 6.00 ng/mL. The spiked recoveries ranged from 97.9% to 105.5%， and intra- and inter-day precision varied between 3.0% and 5.7%. Tear samples from five patients with dry eye and nine healthy volunteers were analyzed using this method. In the dry eye group， the average concentrations of vitamin A and vitamin E were 9.60 （5.13-12.54） ng/mL and 42.00 （31.75-128.00） ng/mL， respectively. In the healthy group， the average concentrations of vitamin A and vitamin E were 18.10 （12.46-21.69） ng/mL and 211.00 （181.00-459.75） ng/mL， respectively. Statistical differences in vitamin A and vitamin E levels between the dry eye group and the healthy group were identified using the Wilcoxon Mann-Whitney test （P<0.05）. This study introduces a straightforward and reliable LC-MS/MS method for the detection of tear VA and VE levels， while also investigating their association with dry eye. This offers a novel reference for understanding the potential risk factors associated with dry eye.
    Keywords:  dry eye; liquid chromatography-tandem mass spectrometry （LC-MS/MS）; tears; vitamin A; vitamin E
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.06017
  41. Plants (Basel). 2026 Feb 26. pii: 696. [Epub ahead of print]15(5):
    Optimization of an Extraction Protocol for Untargeted Metabolomics of Vitis vinifera L. Leaves.
    Laura Sánchez-Ochoa, Teresa Garde-Cerdán, Itziar Sáenz de Urturi, Miriam González-Lázaro, Eva P Pérez-Álvarez.
      Viticulture faces increasing challenges due to the susceptibility of Vitis vinifera L. to biotic and abiotic stresses, which trigger defense responses involving the synthesis of secondary metabolites. Untargeted metabolomics has become a powerful tool to explore these metabolic changes; however, the efficiency and reproducibility of metabolomic studies strongly depend on the extraction protocol used. Current literature shows variability in sample handling, solvent composition, and extraction conditions. This study aimed to optimize an extraction protocol for secondary metabolites in grapevine leaves to ensure high recovery of compounds relevant to untargeted metabolomics. Leaves of Vitis vinifera L. cv. Tempranillo were collected, pooled, frozen, and ground under liquid nitrogen. A factorial design was used to evaluate the effects of sample mass, sample-to-solvent ratio, and solvent type on extraction efficiency. Extracts were analyzed using UHPLC-QTOF-MS in both positive and negative ionization modes, and multivariate statistical tools (PCA and OPLS-DA) were used to identify discriminant metabolites. Optimal extraction was achieved using 750 mg of leaf powder, a sample-to-solvent ratio of 100 mg/Ml, and methanol 80% acidified with 0.1% of formic acid. This protocol maximizes the recovery of metabolites and provides a robust basis for future untargeted metabolomics studies of grapevine responses.
    Keywords:  UHPLC-QTOF-MS; extraction; grapevine; ionization mode; leaf metabolites; untargeted metabolomics
    DOI:  https://doi.org/10.3390/plants15050696
  42. Bioanalysis. 2026 Mar 09. 1-7
    Development and validation of an ultra-sensitive UPLC-MS/MS method for quantification of monomethyl auristatin E in cynomolgus monkey plasma: application to pharmacokinetic study.
    Bing Zhang, Lei Zhang, Yanfeng Liu, Weimin Hu, Rui Diao, Lili Xing, Yi Tao, Liang Shen.
       INTRODUCTION: Antibody-drug conjugates (ADCs) represent a cutting-edge approach in cancer therapy, with monomethyl auristatin E (MMAE) frequently used as a payload in ADC development. We have established a novel bioanalytical method characterized by high sensitivity, accuracy, and efficiency for quantifying MMAE in cynomolgus monkey plasma.
    METHODOLOGY: MMAE was extracted using liquid-liquid extraction (LLE) and quantified by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Results: The method exhibited excellent linearity across a concentration range of 5 to 3000 pg/mL (r ≥ 0.99) in cynomolgus monkey plasma, it fulfilled precision (CV ≤ 15%, 20% for lower limit of quantification (LLOQ)) and accuracy (83.98-112.75%) criteria according to meeting validation requirements per regulatory bioanalytical validation guidelines.
    CONCLUSION: This validated method is instrumental in exploring the in vivo pharmacokinetic profile of ADC and elucidating their exposure-response dynamics.
    Keywords:  Antibody-drug conjugates; LC-MS/MS; bioanalysis; method validation; monomethyl auristatin E; pharmacokinetics
    DOI:  https://doi.org/10.1080/17576180.2026.2641573
  43. Rapid Commun Mass Spectrom. 2026 May 30. 40(10): e70049
    Efficient and Simultaneous LC-MS/MS Detection of N-Nitroso and Di-Nitroso Leucovorin in Leucovorin Drug Products: Method Development and Validation.
    Jaya Yalamanchili, Himavathi Ganja, Suresh Salakolusu.
       RATIONALE: The occurrence of complex nitrosamines, namely, N-nitroso drug substance related impurities (NDRSI) in pharmaceuticals is of great concern due to their potential carcinogenic and mutagenic properties. This creates the need for more rigid and more specific methods of analysis to determine NDSRI levels in pharmaceutical products. The main analysis problem is that it is difficult to separate these impurities and the active pharmaceutical ingredient by chromatography, which is due to their structural similarity. This study describes how a new method of measuring two specific impurities in the Leucovorin drug product, N-nitroso leucovorin and di-nitroso leucovorin, was developed and validated.
    METHODS: Quantification was done by selective reaction monitoring by a positive electrospray ionization source. The required separation was achieved with the use of a reverse-phase C-8 column and gradient-elution in the methanol and water solutions, both adjusted with 0.1% formic acid.
    RESULTS: Linearity of the method was established in a range of 0.1 to 1.5 ng/mL, which is effectually 10% to 150% of its necessary regulatory limit. The procedure had a detection limit of 0.05 ng/mL and a quantitation limit of 0.1 ng/mL. Additionally, it also demonstrated excellent specificity, precision, linearity, accuracy, and robustness when dealing with the two impurities.
    CONCLUSIONS: This methodology will therefore offer a crucial solution to drug manufacturers to track and control N-nitroso leucovorin and Di-nitroso leucovorin concentrations, which will have a positive impact on regulatory compliance and patient safety.
    Keywords:  leucovorin; liquid chromatography–tandem mass spectrometer (LC‐MS/MS); nitroso drug substance related impurities (NDSRIs); selective reaction monitoring (SRM)
    DOI:  https://doi.org/10.1002/rcm.70049
  44. Glycobiology. 2026 Mar 09. pii: cwag014. [Epub ahead of print]
    N-glycan mass spectrometry imaging - a valuable tool for biomarker discovery and validation.
    Anand S Mehta, Peggi M Angel, Richard R Drake.
      Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables spatially resolved detection of diverse biomolecules directly from tissues, supporting pathology-guided and unbiased biomarker discovery. By maintaining spatial context, MALDI-MSI overcomes key limitations of bulk omics approaches, which obscure the cellular origins of analytes, particularly in heterogeneous tumors containing abundant normal tissue or extracellular matrix. Complementary biofluids such as serum, urine, saliva, or cerebrospinal fluid are valuable for biomarker validation yet similarly lack spatial information when used as primary discovery materials. Integrating spatially defined diseased tissues with matched proximal biofluids therefore represents a powerful strategy for biomarker development. N-glycan mass spectrometry imaging (N-glycan MSI) has emerged as a versatile platform for characterizing N-glycan distributions in tissues and for quantifying glycan compositions in cells, antibodies, and biofluids. This method relies on on-tissue application of peptide N-glycosidase F (PNGase F) to liberate N-linked glycans, which are subsequently detected by MALDI-MSI to generate micron-scale, two-dimensional glycan maps. The workflow is highly modular, accommodating tissues, cultured cells, arrays, membranes, and isotopic or label-free quantification approaches. Recent advances also include the use of specialized glycosidases to resolve glycan isomers. This review highlights established and emerging N-glycan MSI strategies for biomarker discovery and validation, with applications in liver, brain, breast, and other clinically relevant tissues.
    Keywords:  MALDI-MSI; biomarkers; cancer; disease
    DOI:  https://doi.org/10.1093/glycob/cwag014
  45. J Sep Sci. 2026 Mar;49(3): e70390
    Simultaneous Quantification of Eight Chiral and Achiral Components in Notopterygii Rhizoma et Radix Extract and Rat Plasma Based on Chiral Stationary Phase-HPLC-MS/MS.
    Hongfei Wu, Luhuan Tang, Zihang Xu, Yanyan Chen, Jingyao Xia, Mengjiao Zhou, Yehan Zhu, Yaqi Yao.
      A chiral stationary phase-high performance liquid chromatography-tandem mass spectrometry (CSP-HPLC-MS/MS) approach was developed and validated for the first time to quantify quantification of eight components: the enantiomers of three chiral components notopterol, oxypeucedanin hydrate, oxypeucedanin (in total six configurations), and achiral components nodakenin, imperatorin, isoimperatorin, bergapten, and ferulic acid, in Notopterygii Rhizoma et Radix. By adjusting the types of chiral stationary phase and the composition ratio of the mobile phase, methanol-acetonitrile (75:25, v/v) was selected as the mobile phase (flow rate 0.5 mL/min), and three chiral components notopterol, oxypeucedanin hydrate, and oxypeucedanin were successfully separated into their enantiomers with Chiralpak IG. The method was applied to analyze both plant extracts and rat plasma samples. A considerable variation in content was observed among the eight components in the plant extracts, with their individual concentrations covering a wide range from 1.04 to 20 400 µg/mL (see Section 3 for details). Similarly, their plasma concentrations also spanned from 0.2 to 262.76 ng/mL. The results demonstrated significant differences in the contents of the components. Notably, the chiral components exhibited marked differences between their enantiomers, suggesting that chiral components should be considered in the quality assessment and control of natural products.
    Keywords:  Notopterygium; chiral stationary phase; enantiomer separation; multicomponent quantification
    DOI:  https://doi.org/10.1002/jssc.70390
  46. Molecules. 2026 Feb 25. pii: 770. [Epub ahead of print]31(5):
    Simultaneous Analysis of Biomarkers in Human Hair for Evaluating Chronic Tobacco Smoke Exposure and Stress/Relaxation Using Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry.
    Hiroyuki Kataoka, Akiko Tsuzaki, Sae Kitagawa, Kentaro Ehara.
      Tobacco smoke exposure not only increases the risks of lung cancer and cardiovascular disease, but can be a stressor contributing to mental illness. It is important to clarify the relationship between chronic tobacco smoke exposure and mental stress from the perspective of disease prevention. We developed a simple and highly sensitive method for simultaneously analyzing nine biomarkers: nicotine and cotinine (tobacco smoke exposure markers); cortisol, testosterone, and dehydroepiandrosterone (stress-related markers); and serotonin, melatonin, dopamine, and oxytocin (relaxation-related markers). Biomarkers were extracted and concentrated by in-tube solid-phase microextraction with a Supel-Q PLOT capillary, followed by separation and detection within 7 min using liquid chromatography-tandem mass spectrometry on a Discovery HS F5 column. Calibration curves using stable isotope-labeled internal standards showed good linearity (0.005-100 ng mL-1) with detection limits of 0.09-13.5 pg mL-1. Intra-day and inter-day precision had relative standard deviations below 7.2% and 15.5% (n = 6), respectively, with recovery rates of 84.0-108.8%. The automated method requires only ultrafiltration of hair methanol extract, enabling non-invasive pg-level analysis using just a few milligrams of hair. Hair analysis reflects an association between chronic tobacco smoke exposure and stress. This method is effective for analyzing the relationship between long-term tobacco smoke exposure and chronic stress.
    Keywords:  biomarkers; hair; in-tube solid-phase microextraction (IT-SPME); liquid chromatography–tandem mass spectrometry (LC–MS/MS); stress; tobacco smoke exposure
    DOI:  https://doi.org/10.3390/molecules31050770
  47. Anal Chim Acta. 2026 May 08. pii: S0003-2670(26)00232-1. [Epub ahead of print]1398 345282
    An on-tissue parallel d0/d20-meladrazine derivatization approach for fatty acid peroxidation products imaging in adult zebrafish.
    Ji Yang, Yue Sun, Yueming Li, Zhonghua Wang, Zeper Abliz, Zhi Zhou.
       BACKGROUND: Mass spectrometry imaging (MSI) enables the simultaneous acquisition of spatial distribution, abundance, and structural information of multiple molecules in biological tissues, yet it faces inherent challenges in detecting poorly ionizable or low-abundance analytes. Fatty aldehydes (FALs) and oxygenated fatty acids (oxo-FAs), key subclasses of fatty acid peroxidation products (FAPPs), are of profound physiological and pathological significance. However, their mass spectrometric detection remains a formidable challenge, particularly for in situ desorption and analysis in biological tissue sections.
    RESULTS: A novel hydrogel-assisted on-tissue chemical derivatization (HCD) approach was developed, employing d0/d20-meladrazine parallel labeling coupled with airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI). Through systematic optimization of hydrogel type, reaction time, reaction temperature, and concentration of the derivatization reagent, 313 FAPPs in adult zebrafish were recognized and annotated, including 176 FALs and 137 oxo-FAs. Notably, the derivatization process was performed at 4 °C with a low reagent concentration of 2 mg/mL, which is effective and more compatible with biological sample integrity. Furthermore, custom written R scripts were developed to enable rapid identification and matching of isotopically labeled peak pairs corresponding to derivative product ions. Applying this method to zebrafish whole-body sections yielded spatial distributions of 237 FAPPs. Further application to Congo red (CR)-exposed zebrafish visualized FAPPs dysregulation, indicating CR-induced oxidative stress-mediated damage in the intestine, liver and brain.
    SIGNIFICANCE: This study presents an effective tool for in situ visualization of FAPPs in adult zebrafish, and its application in water pollutant exposure research provide direct evidence of the neurotoxicity and hepatotoxicity of CR. It has broad potential for adaptation to other biological tissues and facilitates elucidating the toxicity mechanisms of environmental toxins or disease mechanisms relating to oxidative stress.
    Keywords:  Adult zebrafish; Fatty acid peroxidation products; Isotope labelling; Mass spectrometry imaging; On-tissue chemical derivatization
    DOI:  https://doi.org/10.1016/j.aca.2026.345282
  48. Analyst. 2026 Mar 11.
    Separating O-desmethylvenlafaxine and tramadol enantiomers using two-dimensional chiral LC × DMS mass spectrometry.
    A Dawson McLachlan, Rashne Vakharia, Emir Nazdrajić, Diana M Cárdenas-Soracá, Leslie M Bragg, Mark R Servos, W Scott Hopkins.
      Tandem mass spectrometry relies on unique parent-to-product transitions for selective analysis. For sets of isomers or isobars that have identical behaviors in multiple separation dimensions (e.g., LC retention, m/z), quantitation is challenging owing to feature convolution. For example, recent environmental analysis of the enantiomers of O-desmethylvenlafaxine (ODV), an anti-depressant manufactured in a racemic mixture, identified tramadol (TRA, a racemic painkiller) as a co-eluting interference. Here, we demonstrate that differential ion mobility spectrometry (DMS) coupled with chiral LC-MS2 can be used to separate and quantify the enantiomers of ODV and TRA. This method was applied to six wastewater influent samples from an Ontario municipal wastewater plant, where the sum of the enantiomeric concentrations was statistically identical to the racemic concentrations observed on reverse-phase LC-MS2 (t-test, α = 0.05, p-value = 0.26 for ODV and p-value = 0.47 for TRA). We also identify low-intensity product ions specific to ODV that enable isolation and quantitation via chiral LC-MS2 alone, albeit at a relatively high limit of quantitation (LOQ) in comparison to the most intense MRM transition (m/z 264 → 58). Using our chiral (LC × DMS)-MS2 method, the instrumental LOQ of each enantiomer of TRA was determined to be 0.67 ng mL-1 and 5.0 ng mL-1 for the enantiomers of ODV.
    DOI:  https://doi.org/10.1039/d6an00119j
  49. J Chromatogr A. 2026 Mar 07. pii: S0021-9673(26)00217-7. [Epub ahead of print]1774 466887
    Feed injection-enabled reversed phase liquid chromatography for simplified analysis of lipophilic drugs and formulations.
    Daniel Foshag, Stephan Buckenmaier, Patrik Petersson.
      Strong sample solvents and lipidic excipients hamper reversed‑phase (RP) liquid chromatography (LC) analysis of lipid‑based pharmaceutical formulations by causing solvent mismatch, resulting in peak distortion and restricting the applicable injection volume, which ultimately reduces the sensitivity to detect impurities. In this work, we evaluated feed injection (FI) under gradient conditions for the analysis of sesame oil-based formulations dissolved in ethyl acetate (EtOAc) and integrated FI with two‑dimensional LC coupled to mass spectrometry (2D‑LC/MS) for impurity profiling. Findings were compared to conventional flow‑through injection (FTI) and sandwich injection, across injection volumes of 1-40 µL. FI at low feed flow fractions (≤ 5 % of mobile phase flow rate) provided ≥ 1:20 online dilution, sustaining column head focusing, and preserving peak shape and linearity across the full injection range. In contrast, higher feed flow fractions (≥ 20 % of the mobile phase flow rate, ≤ 1:5 dilution) resulted in severe fronting peaks and non-linear ultraviolet (UV) detector response. Finally, the 2D transfers resolved matrix‑ and Active Pharmaceutical Ingredient (API)‑related impurities without laborious offline reconstitution or dilution. This work extends the application field of FI from analyte diluents to complex lipid matrices, offering a robust, scalable workflow for pharmaceutical formulation analysis.
    Keywords:  2D-LC; Ethyl acetate (EtOAc); Feed injection; Lipid-based formulation; Online dilution; Sesame oil
    DOI:  https://doi.org/10.1016/j.chroma.2026.466887
  50. Se Pu. 2026 Mar;44(3): 329-337
    [Analysis of urine biomarkers in urothelial carcinoma based on untargeted metabolomics].
    Sai Liu, Mo Wang, Wei Wang.
      Urothelial carcinoma （UC） is a globally prevalent malignancy lacking robust non-invasive biomarkers. Metabolic reprogramming is a recognized cancer hallmark. Untargeted metabolomics enables high-throughput and unbiased analysis of bodily fluids， offering a promising approach for discovering novel biomarkers in UC. This investigation employs untargeted metabolomic profiling to detect novel urinary biomarkers in UC cohorts. The analytical strategy prioritizes tumor-associated metabolic perturbations through pathway-centric characterization of dysregulated biochemical networks. This study systematically characterizes differential metabolites and associated pathway dysregulations in UC cohorts. The approach seeks to establish a reliable metabolic signature with diagnostic and prognostic value. The findings are expected to advance the development of novel clinical tools. UC biomarkers should optimally integrate preclinical identification， treatment response tracking， and precision-tailored interventions. This investigation provides a methodological framework for exploring cancer metabolism in UC. And it offers evidence-based insights to support translational research and precision medicine initiatives in oncology. This study was conducted at Beijing Chao-Yang Hospital， Capital Medical University， between January and December 2020. A total of 60 urine specimens were consecutively collected. They comprised 30 histologically confirmed UC patients and 30 healthy controls with normal urinalysis findings. Clinical data was prospectively collected via structured case report forms. It encompassed baseline demographics， comorbidities， anthropometric and behavioral factors， and UC pathological parameters. All urine samples were collected prior to invasive procedures. And they were labeled， snap-frozen in liquid nitrogen， and stored at -80 ℃ until analysis. Metabolic profiling was performed using a quadrupole-orbitrap high resolution mass spectrometer equipped with a heated electrospray ionization source. Mass spectrometric data processing was performed using Progenesis QI software. Data processing followed the following workflow： raw data import， spectral peak alignment， feature extraction， and deconvolution. The Progenesis QI software generated datasets containing retention time， peak intensity， and mass-to-charge ratios. Multivariate signal decomposition enabled independent resolution of adduct species， including protonated and sodium-adducted ions. Quality control measures included elimination of ion features demonstrating intra-batch coefficient of variation >15% across technical replicates. This rigorous preprocessing protocol ensured removal of unstable signals. And it preserved biologically relevant metabolic features for subsequent multivariate analysis. No statistically significant differences were observed in baseline clinical characteristics between UC and healthy control cohorts （P>0.05）. Untargeted metabolomic profiling was performed using liquid chromatography-tandem mass spectrometry （LC-MS/MS）. Principal component analysis （PCA） revealed no distinct cluster separation between groups. This result was potentially attributed to limited intergroup metabolic variations or restricted sample size. To enhance discriminatory capacity， supervised orthogonal partial least squares-discriminant analysis （OPLS-DA） was implemented for feature selection. This approach effectively addressed variable collinearity while minimizing non-biological noise interference. Differentially expressed metabolites were identified through variable importance in projection scores， fold change and adjusted P-values. Metabolic pathway analysis was conducted， incorporating pathway enrichment analysis. This multi-tiered analytical approach systematically prioritized UC-associated metabolic perturbations while controlling for confounding factors in specimen analysis. Supervised OPLS-DA was employed to identify differential metabolites and associated metabolic pathways. Significant urinary metabolic disparities were detected between UC patients and healthy controls. Alterations were observed in L-Histidine， N-Acetyltryptophan， 5'-methylthioadenosine， N-methylnicotinamide， L-octanoylcarnitine， 3-indolehydracrylic acid， N¹，N¹²-diacetylspermine， pantothenic acid and so on （P<0.05）. Pathway enrichment analysis revealed perturbations spanning amino acid metabolism， nucleotide biosynthesis， vitamin cofactor utilization， and carbohydrate processing. The histidine metabolism pathway demonstrated the highest topological impact. It was followed by the arginine biosynthesis pathway， arginine and proline metabolism pathway， and the tryptophan catabolism pathway. Future validation in larger cohorts and mechanistic studies is warranted to confirm their clinical utility. The aberrant pathways may offer novel biomarkers and therapeutic targets， particularly for patients resistant to conventional therapies.
    Keywords:  liquid chromatography-tandem mass spectrometry （LC-MS/MS）; metabolic reprogramming; untargeted metabolomics; urine; urothelial carcinoma （UC）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.05018
  51. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Mar 04. pii: S1570-0232(26)00095-4. [Epub ahead of print]1275 125006
    Targeted serum metabolomics reveals novel metabolic associations between fatty acid and kynurenine metabolism in nonalcoholic fatty liver.
    Miaoyang Hu, Jie Xie, Hongchao Zhang, Xinjing Wang, Linlin Wu, Hongxia Guan, Jun Wang, Jie Cheng, Lei Jiang, Ke Wei, Rong Gao.
      Nonalcoholic fatty liver disease (NAFLD) is fundamentally characterized by dysregulated hepatic lipid metabolism. Recent evidence suggests that peripheral neurotransmitter metabolism may be involved in NAFLD pathogenesis, yet the relationship between neurotransmitter and lipid metabolism remains incompletely understood. This study employed targeted serum metabolomics to simultaneously investigate alterations in the kynurenine (KYN) pathway and lipid metabolism. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified a concurrent reduction in serum levels of KYN pathway metabolites, including KYN, xanthurenic acid (XA), and its precursor tryptophan (TRP), in NAFLD patients. These changes were significantly accompanied by dysregulated levels of palmitic acid (PA), arachidonic acid (AA), and eicosapentaenoic acid (EPA). Method validation confirmed analytical reliability, with limit of detection (LOD) of 0.2-5 ng/mL and limit of quantification (LOQ) of 0.5-10 ng/mL for both KYN metabolites and fatty acids. Calibration curves displayed excellent linearity (R2 > 0.995), and both intra-day and inter-day precision was satisfactory, with recovery rates meeting validation criteria. To validate these associations, an HFD-induced NAFLD mouse model was used. Parallel reductions in KYN pathway metabolites and dysregulated fatty acid metabolism were observed in the liver. Logistic regression with false discovery rate (FDR) correction revealed that most KYN metabolite levels varied concordantly with fatty acid levels in mice. In summary, this study provides the first systematic demonstration of concurrent dysregulation of the KYN pathway and lipid metabolism in NAFLD, supported by robust chromatographic-mass spectrometric validation. The observed parallel metabolic disturbances offer new perspectives for therapeutic strategies targeting NAFLD.
    Keywords:  Fatty acid; KYN metabolism; LC-MS/MS; NAFLD; Neurotransmitter
    DOI:  https://doi.org/10.1016/j.jchromb.2026.125006
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