bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2026–11–15
forty-six papers selected by
Sofia Costa, Matterworks
  1. Sensitive LC-MS/MS Method for Simultaneous Quantification of Alectinib and Its Major Metabolite M4 in Limited-Volume Human Plasma.
  2. Optimisation of a dilute-and-shoot UHPLC-MS method for high-throughput urinary metabolomics.
  3. Development and systematic evaluation of an untargeted HILIC‑HRMS metabolomics workflow for multi-matrix human samples analyses.
  4. Determination of Acrylamide in Foods by Liquid Chromatography-Tandem Mass Spectrometry with a Porous Graphitic Carbon Column.
  5. In-silico Tandem Mass Spectral Libraries for the Blood Exposome and Metabolomics Workbench Databases: Application of Gemini Large Language Model.
  6. [Determination of 23 perfluorinated and polyfluoroalkyl substances in eggs by ultra-high performance liquid chromatography-tandem mass spectrometry].
  7. 96-Sample Parallel BioSPME-DART-MS Platform for Rapid Screening of Fentanyl and its Analogues in Biofluids.
  8. Direct Analysis in Real Time-Mass Spectrometry Measurement of Antifungals in Human Serum: A Proof-of-Concept Study.
  9. LC-MS determination of dopaminergic catecholamines in tissues and biofluids using streamlined sample preparation and heterocyclic NHS-ester derivatization.
  10. Enhanced Sensitivity in Free Thyroxine Measurement Using Equilibrium Dialysis and LC-MS/MS Coupled with Offline Anion-Exchange Solid-Phase Extraction.
  11. Next-generation hyphenated analytical technologies in natural product research: From high-resolution separation and structural elucidation to dereplication, metabolomics and drug discovery.
  12. Isotope-Coded Click Chemistry-Enabled Enrichment for Reliable Profiling and Quantification of Carboxyl Metabolites.
  13. Comprehensive and rapid analysis of shea butter extracts by supercritical fluid chromatography-charged aerosol detection-ion mobility high-resolution mass spectrometry.
  14. Micro-flow LC- drift tube IMS-MS with high-resolution demultiplexing for enhanced lipidome coverage of volume-limited biological samples.
  15. Simultaneous quantitation of synthetic cathinone positional isomers in wastewater and urine using liquid chromatography-high-resolution mass spectrometry.
  16. Large-Scale Quantification of Xenobiotics Following Non-Targeted Liquid Chromatography Tandem Mass Spectrometry.
  17. Development and Validation of a UPLC-MS/MS Method for the Determination of Emerging Synthetic, Semi-Synthetic and Related Cannabinoids in Human Hair.
  18. Orthogonal chemical encoding and isotope-coded derivatization enable accurate LC-MS/MS quantification of hydroxy and keto acids in biological samples.
  19. [Online gel permeation chromatography-gas chromatography-triple quadrupole mass spectrometry for determination of 18 polychlorinated biphenyls in serum].
  20. A Multiplex LC-MS/MS Approach for Quantifying Antiepileptic Drugs for Therapeutic Drug Monitoring: Validation of 15 Representative Tests.
  21. [Simultaneous Determination of 21 Etomidate Analogues and Etomidate Acid in Hair by UPLC-MS/MS].
  22. DESI-Enabled Signature-Ion-Guided Natural Product Imaging (DESIGNi) for Rapid Screening of Aromatic Acylated Derivatives in Plant Tissues.
  23. Instrument-Agnostic Machine Learning Framework for Accurate Prediction of Functional Groups from Tandem Mass Spectrometry.
  24. A UHPLC-MS/MS method for the quantification of oxytocin in human saliva.
  25. A Pseudo-Targeted Method for Global Profiling of Cholesteryl Esters by Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring Mass Spectrometry.
  26. Multiclass screening and quantification of drugs and toxicants in whole blood using high-resolution multiple reaction monitoring and information-dependent acquisition under negative electrospray ionization.
  27. Protocol for constructing correlation-based molecular networks from large-scale untargeted metabolomics data.
  28. Single-Cell Mass Spectrometry with Enhanced Coverage of cis-Diol Metabolites via a Reactive Pulled Flowprobe in Static-Zone Enrichment Sampling Mode.
  29. Identification of Geographical Chemical Markers in Neolitsea pallens Using UHPLC-Q-TOF-MS/MS and Multivariate Analysis.
  30. Targeted urine metabolomics for enhanced diagnosis of hypoxic-ischemic encephalopathy in newborns: A pilot study.
  31. Sensitive Quantification of N-Nitroso Dibutylamine Impurities in Tramadol Tablets Using Optimized LC-MS/MS Method.
  32. Ultrasensitive Quantification of Poloxamer 188 in Adeno-Associated Virus Products by All-Ion Fragmentation MS/MS.
  33. A Five-Minute UHPLC-MS/MS Method Facilitates Anthocyanin Biofortification in Maize (Zea mays L.).
  34. Chiral Analysis of 2-Hydroxyglutarate in IDH-mut Glioma Tissue using the Kinetic Method.
  35. Efficient Separation of Disaccharide Isomers Using Ion Mobility Spectrometry by Metal Cation and Anion Assisted Strategy.
  36. Stable Isotope-Assisted Liquid Chromatography Mass Spectrometry Reveals Uptake, Metabolism, and Resolvin Biosynthesis from Docosapentaenoic Acid in Atlantic Salmon (Salmo Salar L.) Hepatocytes.
  37. A Novel Method for the Determination of Ganciclovir Triphosphate in Human Red Blood Cells by LC-MS/MS.
  38. Development of a Rapid and Simple LC-MS/MS Method for the Quantitation of Baricitinib in Human Plasma and Aqueous Humor.
  39. Comparison of Three Urine Normalization Strategies for Mass Spectrometric Analysis in a Pediatric Asthma Model.
  40. Development of a targeted differential mobility spectrometry UHPLC-MS/MS quantitation method for free long-chain and hydroxylated fatty acids.
  41. Hybrid QuEChERS-Micro-Solid Phase Extraction for the Determination of Chloramphenicol in Honey by HPLC-MS/MS: Method Validation and Matrix-Effect Assessment.
  42. Forensic toxicological application of a targeted UPLC-MS/MS method for 15 controlled and prescription-related drugs in human hair: Validation and analysis of 369 anonymized authentic samples.
  43. Spatial Metabolomics Reveals Tissue Specificity and Distribution of Metabolites in Paeonia lactiflora Roots.
  44. AI-Assisted Molecular Structure Prediction from Tandem Mass Spectra: Reliability and Validation.
  45. Quantitative Analysis of Branaplam (LMI070) in Human Plasma by LC-MS/MS in Support of Clinical Trial in Infant Patients With Spinal Muscular Atrophy (SMA).
  46. Metabolomics and lipidomics in the differentiation of CIN 2 and CIN 3: an exploratory pilot study.
  1. Biomed Chromatogr. 2026 Dec;pii: e70658. [Epub ahead of print]40(12):
    Sensitive LC-MS/MS Method for Simultaneous Quantification of Alectinib and Its Major Metabolite M4 in Limited-Volume Human Plasma.
    Reiko Ando Makihara, Makoto Maeda, Ayumu Arakawa, Hitomi S Okuma, Yasushi Goto, Yoshimasa Saito, Hironobu Hashimoto.
      Alectinib is an oral anaplastic lymphoma kinase inhibitor, and M4 is its major pharmacologically active metabolite. Simultaneous quantification of alectinib and M4 in limited-volume plasma is important for pediatric clinical studies, particularly when concentrations are low or exceed the calibration range. An LC-MS/MS method was developed and validated for simultaneous quantification of alectinib and its major metabolite, M4, in human plasma. Alectinib and M4 were extracted from 20 μL of plasma by protein precipitation and quantified using stable isotope-labeled internal standards. Calibration ranges were 0.100-20.0 ng/mL for alectinib and 1.00-200 ng/mL for M4, with lower limits of quantification of 0.100 and 1.00 ng/mL, respectively. Full validation demonstrated acceptable selectivity, linearity, matrix effects, dilution reproducibility, stability, and carryover. Inter-day accuracy (RE) ranged from -9.9% to 5.1%, and inter-day precision (CV) was ≤ 8.3% for both analytes. Partial validation after analytical column replacement also met predefined acceptance criteria. The method was successfully applied to plasma samples obtained from one patient enrolled in an investigator-initiated clinical trial of alectinib, with both alectinib and M4 remaining quantifiable up to 72 h after a single oral dose. This sensitive LC-MS/MS method supports the simultaneous quantification of alectinib and M4 in limited-volume pediatric clinical study samples.
    Keywords:  active metabolite; anaplastic lymphoma kinase inhibitor; bioanalytical method validation; pediatric pharmacokinetics; small‐volume plasma
    DOI:  https://doi.org/10.1002/bmc.70658
  2. Metabolomics. 2026 Nov 13. pii: 176. [Epub ahead of print]22(6):
    Optimisation of a dilute-and-shoot UHPLC-MS method for high-throughput urinary metabolomics.
    Patricia Kelly, Caroline Kerbiriou, Bernadette White, Konstantinos Gkikas, Kelly Stewart, Gillian Farrell, Konstantinos Gerasimidis, Nicholas J W Rattray.
       INTRODUCTION: Urinary metabolomics using liquid chromatography-mass spectrometry (LC-MS) holds great potential for biomarker discovery and clinical application. Large-scale studies and clinical trials require rapid, scalable methods. However, existing urine extraction and LC-MS approaches lack standardisation and clear recommendations for high-throughput use.
    OBJECTIVES: To optimise a dilute-and-shoot urinary metabolite extraction and untargeted ultra-high-performance LC-MS (UHPLC-MS) method suitable for large-scale studies, and to demonstrate its performance in a clinical cohort.
    METHODS: We systematically optimised eight parameters: extraction solvent, dilution factor, chromatographic gradient, injection volume, flow rate, gradient curve factor, full scan automatic gain control (AGC) and MS/MS AGC. Performance was assessed by the number of metabolites detected, peak quality, creatinine performance and peak quality factors (PQFs). The optimised method was applied to 1094 urine samples (862 clinical samples plus QCs) from healthy controls and patients with inflammatory bowel disease (IBD). We compared three normalisation strategies for their effect on data precision: no normalisation, creatinine normalisation and probabilistic quotient normalisation (PQN).
    RESULTS: The optimised method used a C18 column in positive ionisation mode. The mobile phases were water and acetonitrile, each with 0.1% formic acid (v/v). Data acquisition took 6.5 min, and the total run time was 10 min including a cleaning step. In the IBD cohort, over 1500 metabolites were putatively annotated in 182.3 h of instrument time. This saved over 91 h compared with the previously used method.
    CONCLUSIONS: This optimised method provides rapid, scalable untargeted urinary metabolomics suitable for large clinical studies and trials.
    DOI:  https://doi.org/10.1007/s11306-026-02542-1
  3. Metabolomics. 2026 Nov 13. pii: 178. [Epub ahead of print]22(6):
    Development and systematic evaluation of an untargeted HILIC‑HRMS metabolomics workflow for multi-matrix human samples analyses.
    Camillo Morano, Lucia Centofanti, Sara Penati, Sofia Vanerio, Linda Montavoci, Sara Casati, Rita Paroni, Michele Dei Cas.
       INTRODUCTION: Untargeted metabolomics based on LC-HRMS is widely used to investigate metabolic alterations in biological systems. However, comprehensive metabolome characterization remains challenging due to the chemical diversity of metabolites and the influence of chromatographic and sample preparation conditions on analytical performance.
    OBJECTIVES: This study aimed to develop and systematically optimize an untargeted HILIC-HRMS workflow for broad and reproducible detection of polar metabolites across multiple human biological matrices, including serum, urine, and hair.
    METHODS: Chromatographic conditions were optimized in positive and negative ionizations using representative mixtures of amino acids and organic acids. Extraction procedures were evaluated using eight solvent systems and deuterated internal standards to assess recovery, reproducibility, and metabolite coverage. The optimized workflow was applied to serum, urine, and hair samples collected from 24 healthy volunteers. Metabolite annotation was performed through spectral matching using a commercial HRMS/MS library enriched with an in-house library.
    RESULTS: The optimized method employed ammonium formate/formic acid-based mobile phases in positive ionization, ammonium bicarbonate-based conditions in negative ionization, and methanol as the extraction solvent. The workflow demonstrated robust analytical performance across all matrices, identifying 118 metabolites in serum, 196 in urine, and 166 in hair. Twelve metabolite superclasses were detected, with 65 metabolites shared among all matrices. Comparable chemical class distributions indicated broad metabolome coverage with minimal class-specific analytical bias.
    CONCLUSION: The proposed HILIC-HRMS workflow provides a robust, reproducible, and transferable platform for untargeted metabolomics. Its applicability across serum, urine, and hair makes it suitable for large-scale clinical and translational studies focused on polar metabolite profiling.
    Keywords:  Metabolome coverage; Multi-matrix analysis; Polar metabolomics; Untargeted metabolomics
    DOI:  https://doi.org/10.1007/s11306-026-02549-8
  4. Molecules. 2026 Nov 04. pii: 3981. [Epub ahead of print]31(21):
    Determination of Acrylamide in Foods by Liquid Chromatography-Tandem Mass Spectrometry with a Porous Graphitic Carbon Column.
    Huijiao Chen, Lingang Chen, Weiping Xie.
      A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was established for the determination of acrylamide in foods using a porous graphitic carbon (PGC) column for separation. Samples were spiked with an isotopically labeled internal standard, extracted with acetonitrile-water, purified using a mixed-mode cation-exchange (MCX) solid-phase extraction cartridge, and then separated on a PGC column. By employing a single-step pass-through MCX clean-up, the inherent anti-interference capacity of the PGC column in complex matrices was evaluated. Methanol and 0.1% formic acid in water served as the mobile phase, and detection was performed in positive electrospray ionization mode with multiple reaction monitoring (MRM). Method validation was conducted using meat products and coffee samples, and the method was further applied to eight sample categories (n = 68), including potato chips, biscuits, instant coffee, and local specialty foods from southern Fujian. Acrylamide exhibited good linearity over the concentration range of 10-500 µg/L, with correlation coefficients of 0.9994-0.9998. The limit of detection (LOD) was 0.81-1.38 µg/kg, and the limit of quantification (LOQ) was 2.70-4.61 µg/kg. The mean spiked recovery was 94.2-103.4%, with a relative standard deviation (RSD) of 0.8-5.4%, and the matrix effect was 60.6-76.5% for the PGC column, compared with 46.8-51.3% for the C18 column. A paired t-test showed that the PGC column exhibited a significantly higher matrix effect (ME) value (i.e., less signal suppression) than the C18 column (p < 0.05). Owing to the strong retention characteristics of the PGC column for polar compounds, the method effectively improves chromatographic separation and reduces co-eluting interferences. It provides a practical approach for reducing matrix interference and improving sensitivity in matrices where interferences are well-controlled (e.g., meat products), while effectively overcoming the overestimation of acrylamide in highly complex matrices like coffee, with a residual matrix effect corrected by the isotope-labeled internal standard specifically within these validated matrices.
    Keywords:  acrylamide; food safety; liquid chromatography–tandem mass spectrometry; matrix effect; porous graphitic carbon column
    DOI:  https://doi.org/10.3390/molecules31213981
  5. bioRxiv. 2026 Oct 25. pii: 2026.10.20.760278. [Epub ahead of print]
    In-silico Tandem Mass Spectral Libraries for the Blood Exposome and Metabolomics Workbench Databases: Application of Gemini Large Language Model.
    Tuan Nguyen, Dinesh Barupal.
      Human body contains endogenous metabolites, nutrients and xenobiotics chemicals. These chemicals can be detected in a biospecimen using high-resolution mass spectrometry instruments, yielding metabolomic and exposomic datasets. Mass spectral libraries are critical components of these workflows to annotate experimental data with chemical information. In this data descriptor, we report mass spectral libraries for human-relevant small molecules, that are generated using Google Gemini large-language model. Using an optimized chain-of-thought prompt and instructions, we have generated mass spectra for each compound at high/medium/low energy fragmentation level. Each spectrum was reported in the NIST MSP text format for easy to use by metabolomics software. These libraries cover 91,231 unique 2D structures from the Blood Exposome Database and the Metabolomics Workbench data repository. Technical validation was shown by comparing the Gemini-generated spectra against NIST 23 mass spectral database, which demonstrated 55% accuracy. LLMs represent a useful and accessible tool for expanding in-silico spectral libraries to annotate compounds in the HRMS data for human biospecimens and may accelerate the compound annotation in metabolomics and exposomics. Both mass spectral libraries are available at https://zenodo.org/records/20102592 .
    DOI:  https://doi.org/10.64898/2026.10.20.760278
  6. Wei Sheng Yan Jiu. 2026 Sep;55(5): 775-794
    [Determination of 23 perfluorinated and polyfluoroalkyl substances in eggs by ultra-high performance liquid chromatography-tandem mass spectrometry].
    Xueli Yang, Wentao Luo, Yalei Yang, Xuefei Chen, Fang Li.
       OBJECTIVE: To establish a method for the simultaneous determination of 23 perfluorinated and polyfluoroalkyl substances（PFASs）, including perfluoroalkyl carboxylic acids（PFCAs）, perfluoroalkyl sulfonic acids（PFSAs）, and fluorotelomer sulfonic acids（FTSs）, in eggs, using organic solvent extraction, solid-phase extraction（SPE） cleanup, and ultra-high performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）.
    METHODS: Chromatographic separation conditions, mass spectrometry detection parameters, SPE sorbents, reconstitution solvent, and syringe filter were optimized to achieve the best recovery and purification efficiency. Egg samples were extracted with acetonitrile, purified using a high-efficiency lipid-removal SPE column（HLB-P/HMR-Lipid）, and separated on a C18（2.1 mm×150 mm, 3 μm） reversed-phase column with a mobile phase consisting of 5 mmol/L ammonium formate in water and methanol. The analytes were detected in negative electrospray ionization（ESI-） mode with multiple reaction monitoring（MRM） and quantified using stable isotope-labeled internal standards.
    RESULTS: The 23 PFASs exhibited good linearity in the range of 0.20-50.0 ng/mL（r>0.990）. The limits of detection（LOD） and limits of quantification（LOQ） were 0.0022-0.0189 μg/kg and 0.0066-0.0567 μg/kg, respectively. The recoveries ranged from 70.2% to 110.6%, with relative standard deviations of 1.2%-8.9%（n=7）. This method was then applied to determine PFASs in 51 commercially available egg samples collected from Xinjiang. A total of five PFASs were detected, with a detection frequency of 33.3% and concentrations ranging from 0.185 to 1.88 μg/kg.
    CONCLUSION: This method is simple, sensitive, selective, and reproducible, making it suitable for the rapid determination of 23 PFASs in eggs.
    Keywords:  egg; perfluorinated and polyfluoroalkyl substances(PFASs); solid phase extraction; ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)
    DOI:  https://doi.org/10.19813/j.cnki.weishengyanjiu.2026.05.011
  7. Anal Chem. 2026 Nov 10. 98(44): 31703-31711
    96-Sample Parallel BioSPME-DART-MS Platform for Rapid Screening of Fentanyl and its Analogues in Biofluids.
    Wei Zhou, Marc Driezen, Janusz Pawliszyn.
      Solid-phase microextraction (SPME) with biocompatible coating material is highly compatible with ambient mass spectrometry (AMS), such as direct analysis in real time (DART), for improving sensitivity and reducing matrix effects. However, the practical throughput of SPME-DART-MS remains limited by the mismatch between parallel sample preparation and device-to-source transfer. Here, we introduce a 96-sample parallel BioSPME-DART-MS platform that integrates automated SPME extraction, 96-channel liquid transfer, and a matched 96-position DART mesh interface. This design enables 96 biological samples to be processed in a plate-based format, greatly improves workflow transferability. Thin HLB/PAN-coated SPME blades were used to provide bio-compatible extraction and cleanup from whole blood, plasma, and urine. After desorption in 200 μL of solvent, only 5 μL of the extract was transferred to the DART mesh for rapid MS/MS screening, while the remaining solution was retained for sequential LC-MS/MS confirmation without additional sample preparation. The system enabling an average analysis time of approximately 40 s per sample. Using six fentanyl and its analogues as model analytes, the BioSPME-DART-MS method achieved good sensitivity across the three biofluids, with limits of detection of 0.10-0.50 ng mL-1 in whole blood, 0.03-0.20 ng mL-1 in plasma, and 0.01-0.20 ng mL-1 in urine. In addition, a comparative study of absolute matrix effects revealed that chromatography-free DART-MS and blade spray-MS remained susceptible to ion suppression, whereas LC-ESI-MS showed negligible matrix effects. This platform establishes a high-throughput, two-tier SPME-MS workflow that combines rapid ambient MS screening with chromatography-based confirmation.
    Keywords:  direct analysis in real time; fentanyl; high throughput; rapid screening; solid-phase microextraction
    DOI:  https://doi.org/10.1021/acs.analchem.6c05559
  8. J Mass Spectrom Adv Clin Lab. 2026 ;39(1): x6vfj859
    Direct Analysis in Real Time-Mass Spectrometry Measurement of Antifungals in Human Serum: A Proof-of-Concept Study.
    Julie A Ray, Stephen D Merrigan, Preejith Vachali, Terry L Bates, Zahuindanda Aventura, Samuel Putnam, Gwendolyn McMillin, Mark A Fisher, Kamisha L Johnson-Davis.
       INTRODUCTION: Direct analysis in real time (DART) is a soft ionization technique that uses a heated gas such as helium, argon, or nitrogen to generate a cascade of ionized atmospheric molecules that subsequently ionize analytes in biological samples.
    OBJECTIVES: In this proof-of-concept study evaluating selected antifungals in human serum, 5-fluorocytosine (5FC), itraconazole (ITR), hydroxyitraconazole (OH-ITR), isavuconazole (ISV), ketoconazole (KETO), fluconazole (FLU), and posaconazole (POSA) were assessed as DART candidates using a Bruker EVOQ® DART-TQ+.
    METHODS: Human serum samples were extracted using protein precipitation or liquid-liquid extraction. Quantification was performed by DART tandem mass spectrometry (DART-MS/MS) with internal standards. Precision and linearity across individual analytical measurement ranges (AMRs) were evaluated with rapid sample acquisition (0.24 min linear scans), and the results were compared with those obtained using validated LC-MS/MS methods.
    RESULTS: Linearity and precision were acceptable for all evaluated compounds. Good agreement with LC-MS/MS was observed for 5FC, ITR, OH-ITR, and FLU, with slopes ranging from 0.875 to 1.112 and correlation coefficients (r) ≥ 0.949. POSA extraction by protein precipitation resulted in high bias; however, liquid-liquid extraction or the use of an alternative ion transition reduced bias to <7%. DART replicates demonstrated coefficients of variation <15%. All samples met ion-ratio tolerance criteria of ±50%.
    CONCLUSION: This proof-of-concept study demonstrates the feasibility of quantitative antifungal measurement in human serum using DART-MS/MS with simple sample preparation. A moderate linear association with bias for POSA was likely attributable to matrix effects. More selective sample preparation may improve DART accuracy. Overall, DART-MS/MS shows promise as a rapid, high-throughput alternative to conventional LC-MS/MS.
    Keywords:  DART-mass spectrometry; Direct analysis in real time; LC-MS; antifungals; liquid-liquid extraction; protein precipitation
    DOI:  https://doi.org/10.21627/x6vfj859
  9. Anal Bioanal Chem. 2026 Nov 10.
    LC-MS determination of dopaminergic catecholamines in tissues and biofluids using streamlined sample preparation and heterocyclic NHS-ester derivatization.
    Tereza Hofmanova, Filip Divis, Martin Miko, Miroslav Lisa, Eva Cifkova, Rudolf Andrys.
      The accurate quantification of endogenous catecholamines within the dopaminergic pathway plays a crucial role in neurochemical and clinical diagnostics. However, this process is severely hindered by rapid analyte oxidation and complex matrix interferences. This study introduces a comprehensive bioanalytical strategy that connects structural chemical modification with streamlined sample processing. First, the influence of various heterocyclic scaffolds on derivatization agent synthesis, chemical stability, chemoselectivity, and mass spectrometric (MS) ionization enhancement was systematically evaluated. This evaluation identified β-CCarb-NHS as the most promising candidate. To complement this chemical platform, a simple sample preparation workflow was developed. Catecholamines were stabilized directly during sample homogenization using 0.02% formic acid (FA), followed by a rapid liquid-liquid extraction (LLE) utilizing water, ACN, and chloroform. This approach achieved effective deproteination and lipid removal while eliminating the need for solid-phase extraction (SPE) or time-consuming solvent evaporation. Utilizing deuterium-labeled internal standards throughout, the integrated method was fully validated for 11 target catecholamines across mammalian brain tissue, human serum, and urine. The workflow demonstrated excellent accuracy (86-116%), precision (<15%), and recoveries (>99%), yielding competitive limits of detection (3-217 nM) from a minimal 1 µL injection volume. Finally, the practical utility of the protocol was demonstrated by successfully profiling endogenous baseline levels in real human serum, urine, and river nutria brain tissue. The simple, robust, and cost-effective nature of this workflow makes it ideal for the analysis of large sample sets.
    Keywords:  Catecholamines; Derivatization; LC-MS; Sample preparation; β-Carboline
    DOI:  https://doi.org/10.1007/s00216-026-06908-1
  10. J Mass Spectrom Adv Clin Lab. 2026 ;39(1): 1-17
    Enhanced Sensitivity in Free Thyroxine Measurement Using Equilibrium Dialysis and LC-MS/MS Coupled with Offline Anion-Exchange Solid-Phase Extraction.
    Julie A Ray, Kelly Doyle.
      INTRODUCTION: According to the free hormone hypothesis, only the unbound hormone fraction exerts biological effects. Diagnosis and monitoring of thyroid disorders often require the measurement of free thyroxine (fT4) by equilibrium dialysis, which is considered the gold-standard method for separating free hormones from biological samples. OBJECTIVES: Our objective was to reduce complexity and enhance the sensitivity of a liquid chromatography-tandem mass spectrometry method with online solid-phase extraction (SPE) for measuring fT4. METHODS: Serum aliquots of 200 µL were subjected to equilibrium dialysis. Post-dialysis, increases in analytical sensitivity and specificity were achieved using weak-anion-based SPE prior to liquid chromatographic separation with subsequent analyte detection by a high-sensitivity SCIEX Triple Quad™ 7500 mass spectrometer. RESULTS: Lower and upper limits of quantitation (LLOQ and ULOQ) were 0.1 and 10 ng/dL, respectively. Intra- and inter-day precision evaluated using four matrix-matched quality control samples (0.15, 2.09, 5.10, and 7.94 ng/dL) indicated <10% imprecision, showing excellent agreement with the previous validated method (slope = 1.022, r = 0.9927, bias = 4.01%, n = 123). Chromatograms exhibited a substantial improvement in the signal-to-noise ratio at the LLOQ. CONCLUSION: A complex liquid chromatographic separation with online trapping-column method was successfully transitioned to a simpler off-line SPE liquid chromatographic separation method. Incorporating SPE desalting and using the improved instrument sensitivity of the SCIEX Triple Quad™ 7500 mass spectrometer significantly improved the performance. A reduced injection volume allowed sample reanalysis, which is beneficial for pediatric samples. This method provides a superior analytical approach for clinical fT4 measurement.
    Keywords:  Thyroid disease; equilibrium dialysis; free hormone; free thyroxine; liquid chromatography–tandem mass spectrometry; solid-phase extraction
    DOI:  https://doi.org/10.21627/v8rdvz16
  11. Talanta. 2026 Nov 05. pii: S0039-9140(26)01574-2. [Epub ahead of print]314 130918
    Next-generation hyphenated analytical technologies in natural product research: From high-resolution separation and structural elucidation to dereplication, metabolomics and drug discovery.
    Alamgir A Dar, Malik Mukhtar, S A Shameem, Nasseb Singh.
      Natural products occupy a unique position at the interface of chemistry, biology and medicine and continue to provide structurally diverse molecules for drug discovery, chemical biology, nutrition and therapeutic development. Their investigation, however, is complicated by the enormous chemical complexity of natural-product extracts, the presence of structurally related metabolites, isomeric and isobaric compounds, wide concentration ranges, matrix effects and the frequent occurrence of previously uncharacterized constituents. Over the past decade, advances in chromatographic separation, high-resolution mass spectrometry (HRMS), tandem mass spectrometry (MS/MS), ion mobility spectrometry (IMS), nuclear magnetic resonance (NMR) spectroscopy and computational analysis have fundamentally changed how these challenges are addressed. High performance liquid chromatography-mass spectrometry (HPLC-MS), HPLC-MS/MS, HPLC-NMR, LC-solid-phase extraction (SPE)-NMR and capillary electrophoresis-mass spectrometry (CE-MS) have evolved from individual hyphenated techniques into integrated analytical workflows capable of linking chemical separation with molecular characterization, dereplication and biological prioritization. In parallel, molecular networking, spectral databases, in silico fragmentation, machine learning and metabolomics platforms have transformed the interpretation of increasingly complex datasets. This review critically examines these developments, emphasizing the complementary information generated by chromatographic, MS and NMR measurements and their integration with computational approaches. Particular attention is given to HRMS/MS, data-independent acquisition, ion mobility, LC-SPE-NMR, molecular networking, spectral-library searching, metabolite annotation confidence and emerging artificial-intelligence-assisted workflows. The review argues that the principal transformation in natural-product analysis is not simply improved instrumental sensitivity, but the emergence of multidimensional and computationally integrated workflows that connect extraction, separation, detection, structural annotation, dereplication, metabolomics and bioactivity-guided discovery. Remaining challenges including incomplete databases, isomer discrimination, annotation uncertainty, reproducibility and the need for orthogonal structure confirmation are discussed, together with future opportunities for automated, multidimensional and FAIR-data-enabled natural-product discovery.
    Keywords:  HPLC-NMR; HRMS/MS; Ion mobility; Molecular networking; Natural products; Natural-product drug discovery
    DOI:  https://doi.org/10.1016/j.talanta.2026.130918
  12. Anal Chem. 2026 Nov 10. 98(44): 31846-31854
    Isotope-Coded Click Chemistry-Enabled Enrichment for Reliable Profiling and Quantification of Carboxyl Metabolites.
    Xin Tao, Min Ren, Tao Zhang, Hou-Kai Li, Jian-Bo Wan.
      Carboxyl-containing metabolites are involved in diverse biological processes and are closely associated with metabolic diseases. However, their sensitive and reliable profiling by liquid chromatography-mass spectrometry (LC-MS) remains challenging because of low endogenous abundance, inefficient electrospray ionization, and substantial interference from complex biological matrices. Here, we developed a click chemistry-enabled enrichment (CCEE) strategy that integrates isotope-coded derivatization with chemoselective enrichment for sensitive and selective profiling and quantification of carboxyl-containing metabolites in biological samples. CCEE markedly improved detection sensitivity and chromatographic performance while effectively reducing matrix interference. The method also exhibited good linearity, reproducibility, and quantitative accuracy. Application of CCEE to human serum enabled broad profiling of the carboxyl submetabolome and revealed metabolic alterations associated with metabolic dysfunction-associated fatty liver disease (MAFLD). Targeted absolute quantification of representative metabolites further validated the observed alterations. Notably, serum bilirubin levels were significantly lower in individuals with MAFLD and strongly correlated with multiple clinical indicators. Overall, CCEE provides a sensitive and selective strategy for profiling and quantifying the carboxyl submetabolome, facilitating the investigation of metabolic dysregulation and the discovery of candidate biomarkers for metabolic diseases.
    Keywords:  bilirubin; carboxyl-containing metabolites; chemoselective enrichment; click chemistry; isotope-coded derivatization
    DOI:  https://doi.org/10.1021/acs.analchem.6c05924
  13. J Chromatogr A. 2026 Dec 06. pii: S0021-9673(26)00928-3. [Epub ahead of print]1790 467602
    Comprehensive and rapid analysis of shea butter extracts by supercritical fluid chromatography-charged aerosol detection-ion mobility high-resolution mass spectrometry.
    Azemina Bajramova, Jesper Larsen, Jeppe Lindegaard Hjorth, Charlotta Turner, Margareta Sandahl.
      Distinguishing high- from poor-quality vegetable oils requires accurate and reliable characterisation. However, current vegetable oil analysis laboratories rely on multiple complementary techniques and methods for the characterisation of different parts of the complex oil, resulting in long analysis times and high costs. This study aims to develop one single comprehensive and rapid method for shea butter, based on supercritical fluid chromatography (SFC) with charged aerosol detection (CAD) and trapped ion mobility time-of-flight (timsTOF) mass spectrometry (MS). The method should yield rapid compound class separation, quantification of each class, and identification of species within the class. Baseline resolution among compound classes in shea butter extracts was achieved with two polar diethyl amine columns coupled in series. The CAD response uniformity of species in the same class was investigated to assess if one standard per class quantification is possible. A CAD evaporation temperature of 25 °C, power function value of 1, together with polynomial regression were essential for achieving high correlation coefficients for the calibration curves and uniform response factors. Identification of species within classes was achieved using multidimensional data from the hyphenated system. This included retention time, m/z, MS/MS spectra, collision cross section values, and signal intensity. In total, 57 features were identified in the shea butter extract, many of which have been previously reported. Precision, quantification limit, and bias were validated and compared to standard methods when possible. The developed SFC-CAD-timsTOF method provides rapid compositional characterization, simplified class-based quantification using few chemical standards, and analytical performance similar to existing standard methods.
    Keywords:  Charged aerosol detection; Shea butter; Supercritical fluid chromatography; Trapped ion mobility time of flight mass spectrometry; Vegetable oil
    DOI:  https://doi.org/10.1016/j.chroma.2026.467602
  14. Talanta. 2026 Nov 09. pii: S0039-9140(26)01573-0. [Epub ahead of print]314 130917
    Micro-flow LC- drift tube IMS-MS with high-resolution demultiplexing for enhanced lipidome coverage of volume-limited biological samples.
    Guillermo Viejo-Palenzuela, Lydia Álvarez, Jorge R Álvarez-Buylla, Héctor González-Iglesias, Marcos Bouza.
      The analysis of volume-limited biological specimens remains a major challenge in lipidomics, requiring analytical platforms with improved sensitivity and structural characterization capabilities. Here, a microflow liquid chromatography ion mobility mass spectrometry (microLC-IMS-MS) platform incorporating high-resolution demultiplexing (HRdm) was developed and benchmarked against conventional ultraperformance liquid chromatography (UPLC) for untargeted lipidomics. Following optimization of the LC hardware and ion source conditions, microLC provided median 2- to 14-fold increases in peak area, up to 10-fold increases in peak height, and approximately 3-fold higher signal-to-noise ratios relative to UPLC. Calibration with 34 isotopically labeled standards spanning eight lipid classes gave detection limits of 0.22-14.3 μg/L (median 1.6 μg/L; 0.80 pg on column) and quantitative ranges covering a median of 2.2 orders of magnitude. Analysis of NIST SRM 1950 plasma enabled the annotation of 130 lipid species compared with 74 by UPLC-MS, representing a 76% increase in lipid coverage. When combined with HRdm, the platform resolved five lipid isomer pairs that remained unresolved under conventional UPLC conditions. The method was further applied to volume-limited biological specimens, enabling the identification of 62 lipids from as few as 1x103 human Müller glia cells and lipid profiling of human vitreous humor samples from a pilot cohort of glaucoma patients and matched controls. Variations in lipid features were observed in an exploratory pilot clinical study, supporting the applicability of the platform to clinically relevant ocular biofluids; given the small cohort, these observations should be regarded as hypothesis-generating. These results establish microLC-HRdm-MS as a sensitive and reproducible platform for lipidomics of volume-limited and structurally complex specimens.
    Keywords:  Bioanalytical applications; High-resolution demultiplexing; Ion mobility mass spectrometry; Lipidomics; Micro-flow liquid chromatography; Volume-limited specimens
    DOI:  https://doi.org/10.1016/j.talanta.2026.130917
  15. Anal Methods. 2026 Nov 09.
    Simultaneous quantitation of synthetic cathinone positional isomers in wastewater and urine using liquid chromatography-high-resolution mass spectrometry.
    Jiaxing Liu, Peng Che, Coryn LLamoza, Marja Lamoree, Isabelle Kohler, Jeroen Kool.
      Over the past decade, an alarming number of new compounds (NPSs) have emerged in the recreational drug market, posing serious threats to both public health and society. Many NPSs are positional isomers or structurally related compounds of illegal substances, which potentially harm public health due to their largely unknown pharmacological and toxicological effects, leading to tremendous demand for powerful and effective NPS screening methods. Synthetic cathinones (SCs) are the second most prevalent class of NPSs in Europe, a class encompassing a large number of isomers, showing the need for adequate analytical approaches to discriminate them. Urine testing is typically used to detect NPSs, with samples obtained through a non-invasive sampling technique. In addition, wastewater analysis provides a complementary method to monitor community-level trends in the use of these rapidly evolving substances. This study presents the development and validation of an analytical method based on solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous separation and quantitation of three sets of synthetic cathinone positional isomers, namely, chloromethcathinone, methylones, and ethylones in both wastewater and urine matrices. An LC-MS/MS method using high-resolution multiple reaction monitoring (MRMHR) acquisition was developed for the analysis of spiked wastewater and urine matrices, and its analytical performance was compared with that of high-resolution selected ion monitoring (HR-SIM). The developed method was validated in terms of selectivity, linearity and range, precision, accuracy, limit of detection, and lowest limit of quantification. Recovery and matrix effects were also assessed. The lower limits of quantification (LLOQs) for all seven compounds were as low as 0.1 ng mL-1 in both matrices. The developed method provides a validated analytical framework for the selective separation and quantitation of synthetic cathinone positional isomers in urine and wastewater matrices.
    DOI:  https://doi.org/10.1039/d6ay00862c
  16. J Am Soc Mass Spectrom. 2026 Nov 06.
    Large-Scale Quantification of Xenobiotics Following Non-Targeted Liquid Chromatography Tandem Mass Spectrometry.
    Maxence Derbez-Morin, Rithi Krishnaraj, Haiyan Ke, Thomas Cummins, Amancio De Souza, Tilman Schramm, Yourae Shin, Ansel Hsiao, Mingxun Wang, Daniel Petras.
      The diversity of anthropogenic organic substances in aquatic environments creates a need for analytical workflows that combine chemical coverage with reliable quantification, in order to assess global chemical pollution. Here, we present an integrated workflow that combines non-targeted and targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) for the analysis of xenobiotics in river and coastal environments. We developed a web-based application, GNPS2-Quant, to convert data-dependent acquisition (DDA) results into scheduled inclusion lists for parallel reaction monitoring (PRM), using precursor m/z values and retention times, thereby accelerating the transfer from non-targeted discovery to targeted quantification within the same instrument platform. Using a quadrupole orbitrap (Exploris 480) instrument and a panel of 142 xenobiotics, we compared the quantification performance of full-scan DDA-MS1 data vs PRM. While PRM provides greater selectivity, DDA-MS1 generally provided greater sensitivity, yielding lower limits of detection for 61 compounds and lower limits of quantification for 61 compounds. Despite these differences, both methods showed comparable quantitative accuracy, with approximately 75% of quality control measurements meeting our precision criteria, which was in a similar range to measurements using multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. Application to Southern California river and estuary samples enabled the quantification of 57 xenobiotics, with strong agreement between methods across sites. While our results highlight the utility of PRM generation from non-targeted LC-MS/MS data, our data also demonstrate that DDA-MS1 alone can provide scalable quantitative performance, avoiding costly and time-consuming reanalysis.
    Keywords:  DDA; DOM; PRM; Quantification; Xenobiotics; non-targeted analysis
    DOI:  https://doi.org/10.1021/jasms.6c00334
  17. Molecules. 2026 Oct 22. pii: 3816. [Epub ahead of print]31(21):
    Development and Validation of a UPLC-MS/MS Method for the Determination of Emerging Synthetic, Semi-Synthetic and Related Cannabinoids in Human Hair.
    Leonardo Romani, Giulio Mannocchi, Lucrezia Stefani, Luigi Tonino Marsella, Roberta Tittarelli.
      Synthetic cannabinoids (SCs) are an evolving group of new psychoactive substances (NPSs), characterized by structural diversity and continuously emerging analogues. Their detection in biological matrices is challenging because of low concentrations, rapid metabolism, and incomplete coverage by routine methods. Hair analysis offers a detection window for retrospective exposure assessment. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous identification and quantification of 26 synthetic, semi-synthetic, and related cannabinoids in human hair and to assess its applicability to authentic forensic samples. The method was validated for linearity, limits of detection (LOD) and quantification (LLOQ), accuracy, precision, matrix effect, recovery and process efficiency, with additional robustness assessments under freeze-thaw and processed-extract conditions, and was then applied to n = 100 hair samples collected from subjects tested positive for Δ9-tetrahydrocannabinol (Δ9-THC) during forensic assessments following driving license suspension. LODs were 1.56-3.13 pg/mg, LLOQs 6.25-12.5 pg/mg, and R2 ≥ 0.992; accuracy and precision met the predefined acceptance criteria, while ME, RE, and PE showed generally consistent mean performance with wider level-specific variability for selected analytes. Target cannabinoids were detected in 23/100 samples, with 14 different compounds identified. MDMB-4en-PINACA, 9R-HHC-O, and ADB-BUTINACA were the most frequently detected compounds. The method enabled sensitive and reliable detection of multiple emerging cannabinoid classes in hair, supporting the continuous updating of targeted analytical panels in response to the evolving NPS landscape.
    Keywords:  UPLC-MS/MS; forensic toxicology; hair analysis; method validation; synthetic cannabinoids
    DOI:  https://doi.org/10.3390/molecules31213816
  18. J Chromatogr A. 2026 Nov 07. pii: S0021-9673(26)00937-4. [Epub ahead of print]1791 467611
    Orthogonal chemical encoding and isotope-coded derivatization enable accurate LC-MS/MS quantification of hydroxy and keto acids in biological samples.
    Yusong Wang, Xueying Wang, Yupei Jiao, Lina Xu, Xiaohui Liu.
      Hydroxy and keto acids are important intermediates in central carbon metabolism and redox regulation, yet their comprehensive quantitative analysis remains challenging because of high polarity, poor chromatographic retention, and substantial concentration variation in biological matrices. Here, we develop an orthogonal chemical encoding strategy coupled with LC-MS/MS for simultaneous and quantitative profiling of hydroxy and keto acids. Keto acids are selectively modified at carbonyl groups using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA), followed by carboxyl-directed derivatization of both keto and hydroxy acids with 3-nitrophenylhydrazine (3-NPH). This sequential labeling approach introduces complementary chemical signatures for distinct metabolite classes, improving keto acid detectability while preserving broad coverage of hydroxy acids. The analytical performance of the method was systematically evaluated, demonstrating wide linear dynamic ranges, subnanomolar detection limits for most analytes, high precision, and minimal matrix effects for 20 hydroxy and keto acids. Furthermore, isotope-coded derivatization using 13C6-3-NPH enabled accurate isotope-based internal standardization without requiring individual isotope-labeled standards for each analyte. The applicability of the method was demonstrated by profiling serum samples from diabetic mice, revealing altered ketone body metabolism, branched-chain amino acid-derived keto acid pathways, and hydroxy/keto acid interconversion. This orthogonal chemical encoding approach provides a robust analytical strategy for quantitative characterization of structurally related polar metabolites in complex biological matrices.
    Keywords:  Hydroxy acids; Isotope-coded derivatization; Keto acids; LC-MS/MS; Orthogonal chemical encoding
    DOI:  https://doi.org/10.1016/j.chroma.2026.467611
  19. Wei Sheng Yan Jiu. 2026 Sep;55(5): 785-794
    [Online gel permeation chromatography-gas chromatography-triple quadrupole mass spectrometry for determination of 18 polychlorinated biphenyls in serum].
    Leyuan Sun, Yan Zhou, Haichuan Chen, Wencheng Cao, Qingyun Cheng, Xiao Liu, Sheng Wen.
       OBJECTIVE: To establish a rapid method for detecting 18 polychlorinated biphenyls（PCBs） in serum using online gel permeation chromatographygas chromatography-triple quadrupole mass spectrometry（GPC-GC-MS/MS）.
    METHODS: Isotopic internal standards were added to 100 μL serum samples. Following extraction with acetone-cyclohexane（3 ∶7,V/V） and dehydration with anhydrous sodium sulphate, the supernatant was concentrated and analysed by GPC-GC-MS/MS. Target compounds were separated using a DB-5MS column（30 m × 0.25 mm, 0.25 μm） for target compound separation. Detection employed multiple reaction monitoring mode with quantification via internal standardisation.
    RESULTS: The 18 PCBs exhibited good linearity within the concentration range of 0.01-5 ng/mL, with correlation coefficients（R2） all exceeding 0.997.The method limits of detection（LOD） ranged from 0.001 to 0.004 ng/mL. Spiked recovery rates for the 18 PCBs ranged from 80.9% to 118.6% at spiking levels of 0.01, 0.1 and 1ng/mL, with relative standard deviations（RSD） of 1.5% to 12.6%（n=6）. Comparing the result with those of human serum standard reference material SRM1958（0.00778-0.473 ng/g） showed that the coefficient of RSD was less than 10% for all 18 PCBs relative to the reference values. This indicates good accuracy and specificity. Application of the established method to 20 human serum samples from a specific region detected all 12 remaining PCBs, with the exception of PCB-81, PCB-77, PCB-123, PCB-114, PCB-126, and PCB-169.Detection rates ranged from 5% to 100%, with detection levels ranging from below the LOD to 0.807 2 ng/mL.
    CONCLUSION: This method demonstrates high precision and accuracy and provides an effective analytical approach for the rapid and sensitive detection of trace polychlorinated biphenyls in serum.
    Keywords:  gas chromatography-triple quadrupole mass spectrometry; online gel permeation chromatography; polychlorinated biphenyls; serum
    DOI:  https://doi.org/10.19813/j.cnki.weishengyanjiu.2026.05.012
  20. J Mass Spectrom Adv Clin Lab. 2026 ;39(1): hs00ns84
    A Multiplex LC-MS/MS Approach for Quantifying Antiepileptic Drugs for Therapeutic Drug Monitoring: Validation of 15 Representative Tests.
    Xiaoying Tang, Ruhan Wei, Emily Chegwidden, Richard Giles, Adam M Kutnick, Drew Payto, Jessica M Colón-Franco.
       INTRODUCTION: Therapeutic drug monitoring of antiepileptic drugs is critical for optimizing clinical outcomes, minimizing toxicity, assessing drug compliance, and managing overdoses and drug interactions.
    OBJECTIVES: We developed and validated an LC-MS/MS method for the quantification of 15 antiepileptic drugs (ethosuximide, primidone, pentobarbital, carbamazepine-10,11 epoxide, pregabalin, gabapentin, zonisamide, lacosamide, rufinamide, felbamate, lamotrigine, topiramate, 10,11-dihydro-10-hydroxycarbamazepine, perampanel, and brivaracetam).
    METHODS: Antiepileptic drugs were extracted from plasma and serum using methanol-based protein precipitation, followed by dilution. A commercial ClinCal® 3-point calibrator was used for all analytes except pentobarbital, which used a 6-point in-house calibrator. Chromatographic separation was achieved using a reverse-phase C18 column with a 7.31 min elution gradient of water and methanol, both containing 2 mM of ammonium acetate.
    RESULTS: All analytes required a 5 µL injection volume, except ethosuximide and pentobarbital, which required 20 µL and 10 µL, respectively, for optimal performance. The method demonstrated excellent linearity for all 15 antiepileptic drugs within their respective concentration ranges, with acceptable selectivity, accuracy (80.0-118.3%), and intraday and interassay precision (CV < 7.4%). Stability studies showed that antiepileptic drugs were stable in serum and plasma for up to 24 h at room temperature, 7 days at 4 °C, and 30 days at -20 °C, and after extraction for 7 days at 4 °C.
    CONCLUSION: This LC-MS/MS method supports routine quantification of 15 antiepileptic drugs in a clinical laboratory using a common sample preparation and chromatography workflow across all measurements. It was operationalized into three instrument methods to accommodate the different injection volume and calibrator requirements.
    Keywords:  LC-MS/MS; antiepileptic drug; clinical laboratory; method validation; therapeutic drug monitoring
    DOI:  https://doi.org/10.21627/hs00ns84
  21. Fa Yi Xue Za Zhi. 2026 Aug 25. pii: 1004-5619(2026)04-0318-10. [Epub ahead of print]42(4): 318-327
    [Simultaneous Determination of 21 Etomidate Analogues and Etomidate Acid in Hair by UPLC-MS/MS].
    Xing Han, Kan Li, Xin Liu, Chao Liu, Yuesen Shi, Qingkun Hu, Mingluo Du, Zhenhua Qian, Xiaobo Wang, Hui Zheng.
       OBJECTIVES: To establish a qualitative and quantitative analytical method for the determination of 21 etomidate analogues and etomidate acid in hair and to investigate its application value in actual forensic cases.
    METHODS: Hair samples were extracted following cryogenic wet grinding. The supernatant was filtered through a 0.22 μm organic membrane filter. The Kinetex® Biphenyl (100 mm×2.1 mm, 1.7 μm) column was used, and gradient elution was performed with methanol and 5 mmol/L ammonium acetate within 15 minutes. The data acquisition was acquired by UPLC-MS/MS with electrospray ionization in positive ion mode, followed by method verification.
    RESULTS: The 21 etomidate analogues and etomidate acid exhibited good chromatographic separation in hair samples. In particular, all eight compounds within three distinct groups of isomers, including propoxate and isopropoxate; butomidate, sec-butomidate, iso-butomidate and tert-butomidate; 1-(methoxycarbonyl)-2-methylpropan-2-yl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate and 1-(methoxycarbonyl)-2-methylpropanyl 1-(1-phenylethyl)-1H-imidazole-5- carboxylate, were effectively separated. The method showed a good linear relationship in the quantitative linear range (r>0.999), with the limits of detection (LOD) ranging from 0.005 to 0.020 ng/mg and the lower limit of quantification (LLOQ) of 0.05 ng/mg. The selectivity, accuracy and precision of the established method met the verification standards. The matrix effects and extraction recoveries showed good reproducibility at different concentration levels, and the quantitative results for processed hair samples remained stable after storage at 4 ℃ for 36 hours. Practical application results showed that etomidate, metomidate, isopropoxate, sec-butomidate, iso-butomidate, TF-etomidate, and ABP-700, and the metabolite etomidate acid could be detected in the hair of abusers, although the content of etomidate acid was relatively low.
    CONCLUSIONS: The method established in this study is simple to perform and provides accurate quantification. It enables the simultaneous determination of etomidate acid, 13 scheduled etomidate analogues, and 8 unscheduled potential derivatives in hair. The analytical scope encompasses alkyl, alkoxy, alkoxycarbonyl, halogen, and sulfhydryl substitutions distributed across the ester side chain, benzene ring, and imidazole ring. It can provide technical support for combating the abuse of etomidate analogue substances.
    Keywords:  etomidate; etomidate acid; forensic medicine; hair; toxicological analysis; ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
    DOI:  https://doi.org/10.12116/j.issn.1004-5619.2026.360203
  22. Anal Chem. 2026 Nov 10. 98(44): 31217-31227
    DESI-Enabled Signature-Ion-Guided Natural Product Imaging (DESIGNi) for Rapid Screening of Aromatic Acylated Derivatives in Plant Tissues.
    Yuchen Xue, Jinmin Liu, Mingju Yao, Xiaohui Zhang, Fei Han, Xiaomeng Shi, Xue Qiao.
      Mass spectrometry imaging (MSI) enables in situ visualization of small-molecule distributions in plants. However, MSI data interpretation often relies on extensive prior chemical knowledge, limiting the rapid screening of specific compound classes. Furthermore, structural analysis via LC/MS typically depends on tandem mass spectrometry, which is not routinely integrated into standard MSI workflows. To address this, we established a desorption electrospray ionization (DESI)-enabled, signature-ion-guided natural product imaging (DESIGNi) method. By acquiring high-resolution fragment ion data using a multi-reflecting time-of-flight (MRT) mass spectrometer, this approach enables rapid visualization of compounds containing specific building blocks based on their diagnostic fragment ions. As a proof of concept, we focused on aromatic acylated derivatives. The method was developed and optimized using the phenylethanoid glycosides in Forsythia fruit, and the accuracy of the resulting distributions was validated by LC/MS. We applied this approach to seven additional medicinal or edible plants and mapped the distribution of phenylethanoid glycosides, phenolic acid oligomers, benzyl ester glycosides, amides, and diarylheptanoids. By tracing signature ions to their corresponding full-scan spectra, MSI enabled the discovery and preliminary identification of rare or potential novel compounds. This study presents a new strategy for rapid visual screening of plant natural products using MSI.
    DOI:  https://doi.org/10.1021/acs.analchem.6c04888
  23. Anal Chem. 2026 Nov 10. 98(44): 30122-30131
    Instrument-Agnostic Machine Learning Framework for Accurate Prediction of Functional Groups from Tandem Mass Spectrometry.
    Connor Beveridge, Prageeth R Wijewardhane, Matthew Muhoberac, Nicolás M Morato, Andreas Kaerner, Li-Kang Zhang, Douglas Richardson, Ayanna Shoulds, Jeffrey R Gilbert, Christopher J Welch, Shane Tichy, Gaurav Chopra.
      Structural elucidation of unknown compounds using tandem mass spectrometry (MS/MS) is an ongoing challenge. Expert chemists will often use common product ion peaks and neutral losses or peak matching software to predict structural aspects from MS/MS spectra, a process which is time consuming and limited by the molecular entries in the database. We introduce an instrument-agnostic machine learning (ML) framework that predicts functional groups directly from MS/MS spectra, eliminating the need for conventional database searches. The ML models are trained on data from different sources, the MassBank of North America (MONA), a public database, and a custom MS/MS database acquired using a single high throughput desorption electrospray ionization (DESI) MS/MS platform to showcase transferability of model predictions independent of instrument acquisition. Additionally, a novel spectral representation is developed for ML use by standardizing the spectra to the precursor ion m/z. We utilized molecular metrics, specifically, average molecular F1 score of 87% (MONA) and 76% (DESI) and average molecular accuracy of 94% (MONA) and 87% (DESI), indicating similar model performance when trained on different databases to validate the ML framework. To further illustrate the robustness and transferability of our ML framework, models trained on DESI-generated data successfully predicted functional groups, using spectral data generated from a blind test set of molecules using two different company laboratories using distinct mass spectrometry instruments (Orbitrap and TOF) operated in positive-ion mode electrospray ionization. We believe that such an approach, where models trained on data from one instrument can reliably make accurate predictions on spectra acquired using different instruments in a database-free manner, will significantly expand the practical applications of ML to mass spectrometry.
    DOI:  https://doi.org/10.1021/acs.analchem.5c05945
  24. Psychoneuroendocrinology. 2026 Nov 06. pii: S0306-4530(26)00338-0. [Epub ahead of print]195 108078
    A UHPLC-MS/MS method for the quantification of oxytocin in human saliva.
    Sara Soloperto, Jacopo Mula, Francesco Chiara, Fabio Favata, Giorgia Menegatti, Elena Cat Genova, Domenico Maiese, Alice Palermiti, Amedeo De Nicolò, Marco Simiele, Rita Pezzati, Antonio D'Avolio, Marco Cantù.
      Given the reported correlation between cerebrospinal fluid (CSF) and salivary oxytocin concentrations, salivary oxytocin has been proposed as a promising peripheral biomarker of central oxytocin activity. To date, oxytocin levels are commonly measured using enzyme immunoassays or radio-immunoassays; however, several challenges related to its accurate quantification have been reported, underscoring the need for a standardized and reliable analytical method. The present study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of oxytocin in human saliva, in accordance with EMA and FDA guidelines for bioanalytical method validation. Oxytocin levels were determined using the standard addition method. The chromatographic separation was performed through a gradient elution of two mobile phases (H2O + 0.1% F.A. and MeOH: DMSO 97:3 (v/v) + 0.1% F.A.) using a Phenomenex Luna Omega 1.6 μm PS C18 100 Å LC Column 50 × 2.1 mm at 40°C with a column thermostat. It was connected to a tandem mass spectrometer, and the chromatographic run lasted 5 min. Additionally, in order to evaluate precision and accuracy of the method, real-world samples from caregivers working with people with dementia were analyzed. Method validation achieved satisfactory results in terms of robustness and reproducibility, especially at higher concentration values, while expected higher variability at lower concentrations was observed. However, real-world samples analysis confirmed the applicability of the method to real biological samples. Overall, the method was able to accurately quantify oxytocin levels in real-world samples, thus demonstrating its applicability in epidemiological studies.
    Keywords:  Liquid chromatography; Oxytocin; Saliva
    DOI:  https://doi.org/10.1016/j.psyneuen.2026.108078
  25. Anal Chem. 2026 Nov 10. 98(44): 30837-30846
    A Pseudo-Targeted Method for Global Profiling of Cholesteryl Esters by Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring Mass Spectrometry.
    Jiarun Huang, Lan Huang, Kaikun Xu, Xinyi Wan, Hanjie Ren, Pei Jiang, Fuchu He, Ya-Li Bai.
      Cholesteryl esters (CEs) play critical roles in diverse physiological processes, yet their comprehensive analysis remains challenging due to the limited availability of authentic standards and the low abundance of many CE species. To address this, we developed a pseudo-targeted screening strategy that integrates virtual database construction with retention time prediction for the global analysis of CEs using optimized liquid chromatography-Zeno high-resolution multiple reaction monitoring (LC-Zeno MRM-HR). Compared to conventional data-dependent acquisition (DDA), our method demonstrated superior sensitivity and specificity. Application of this strategy to biological samples resulted in the identification of 80 CE species. Among these, 18 are reported here for the first time, and 9 were unequivocally validated using authentic standards, underscoring the method's extended coverage and reliable identification accuracy. We further applied this method to analyze CE metabolism in hepatocellular carcinoma (HCC) cells. Pharmacological inhibition of SOAT1 (by avasimibe) and NCEH1 (by JW480) revealed their opposing roles: SOAT1 inhibition depleted CEs, while NCEH1 inhibition promoted their accumulation, consistent with established biology understanding and affirming the utility of our approach. Finally, leveraging the analytical power of this strategy, we conducted a multi-tissue survey across brain, heart, lung, kidney, stomach, fat, and plasma. This revealed distinct, organ-specific CE profiles that correlate with local physiology, establishing a foundational blueprint for tissue-specific CE distribution.
    DOI:  https://doi.org/10.1021/acs.analchem.6c04131
  26. J Chromatogr A. 2026 Dec 06. pii: S0021-9673(26)00929-5. [Epub ahead of print]1790 467603
    Multiclass screening and quantification of drugs and toxicants in whole blood using high-resolution multiple reaction monitoring and information-dependent acquisition under negative electrospray ionization.
    Mengyi Wu, Ke Hu, Zheng Qiao, Junbo Zhao, Liuqing Zhao, Hui Yan, Bin Di, Hang Chen, Ping Xiang.
      Drugs and toxicants exhibiting favorable ionization behavior under negative electrospray ionization (ESI-) represent an important group of analytes in toxicological analysis; however, the development of methods optimized for their analysis in this mode remains limited. This study aimed to establish an ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry strategy for the analysis of drugs and toxicants amenable to ESI- in human blood by integrating high-resolution multiple reaction monitoring (MRMHR) with information-dependent acquisition (IDA). The developed method showed good linearity over concentration ranges of 2-800 ng/mL or 5-1000 ng/mL (r² > 0.994), with limits of detection and quantification of 0.5-2 ng/mL and 2-5 ng/mL, respectively. Precision and accuracy met the acceptance criteria, with acceptable matrix effects and recoveries for most analytes. Among 164 authentic blood samples analyzed, 42 had at least one analyte detected, involving 20 compounds in total. All compounds quantified by MRMHR were detected by IDA, and 9 additional compounds outside the quantitative panel were identified. Of these 42 samples, 28 contained at least one compound beyond the predefined MRMHR targets. This strategy enables reliable quantification and expanded screening of drugs and toxicants detectable in ESI-, providing a complementary analytical strategy for toxicological investigations.
    Keywords:  Blood; Drugs; High-resolution mass spectrometry; High-resolution multiple reaction monitoring; Information-dependent acquisition; Negative electrospray ionization
    DOI:  https://doi.org/10.1016/j.chroma.2026.467603
  27. STAR Protoc. 2026 Nov 10. pii: S2666-1667(26)00609-X. [Epub ahead of print]7(4): 104956
    Protocol for constructing correlation-based molecular networks from large-scale untargeted metabolomics data.
    Huang Lin, Lijun Zhang, Ali Lotfi, Alan Jarmusch, Iris Lee, Adam Kim, James T Morton, Alexander Aksenov.
      Here, we present a computational protocol for constructing correlation-based molecular networks from untargeted metabolomics data using MetVAE, a variational autoencoder-based framework. We describe steps for importing metabolomics features and sample metadata and for adjusting for compositionality, missingness, confounding, and high dimensionality. We then detail procedures for estimating sparse metabolite correlations and exporting GraphML files for network visualization in Cytoscape. By complementing spectral similarity networks, this protocol captures functional relationships reflected in cross-sample correlations.
    Keywords:  Chemistry; High Throughput Screening; Metabolomics; Protocols in Metabolomics and Lipidomics
    DOI:  https://doi.org/10.1016/j.xpro.2026.104956
  28. Anal Chem. 2026 Nov 10. 98(44): 30307-30317
    Single-Cell Mass Spectrometry with Enhanced Coverage of cis-Diol Metabolites via a Reactive Pulled Flowprobe in Static-Zone Enrichment Sampling Mode.
    Ru Ma, Zhihao Zhao, Qian Wu, Ziqin Lu, Yuanxia Lv, Shijiao Chen, Hongmei Lu.
      Single-cell mass spectrometry (MS) faces significant challenges in covering low-abundance and poorly ionizable metabolites, hindering comprehensive single-cell metabolomics studies of disease and life processes. Herein, we proposed a new single-cell MS method based on a pulled flowprobe that enables online static-zone metabolite enrichment and derivatization of cis-diol metabolites, thereby greatly enhancing metabolite coverage. A stationary sample band was observed within the continuously flowing liquid microjunction of the pulled flowprobe when the inner capillary was substantially retracted inside the outer capillary. A larger retraction distance and lower solvent flowrate were found to benefit band formation, and the band served as a static zone to enrich analytes, achieving up to 50-fold signal enhancement. Then, a benzylboronic acid derivatization reagent was introduced into this band, where both the reagent and cis-diol metabolites were enriched to achieve static derivatization, yielding faster reaction kinetics (< 1/4 the time to reach maximum yield) and greatly reduced band diffusion/dilution (peak width: 30 s vs > 100 s) compared with the traditional dynamic online derivatization. Then, the new single-cell MS method with online enrichment and static-zone derivatization was established and enhanced overall metabolite coverage more than two-fold (53 vs 24), and specifically three-fold for cis-diol metabolites from a single cell, compared to the traditional method using dynamic derivatization. Finally, the new single-cell MS method was applied to the neuronal injury study, and six metabolites involved in oxidative stress, myelin damage, and DNA damage pathways were found to be significantly altered between injured and normal cells. These results highlight the advances of our method in expanding the molecular understanding at the single-cell level to uncover disease mechanisms.
    DOI:  https://doi.org/10.1021/acs.analchem.6c03070
  29. Rapid Commun Mass Spectrom. 2027 Jan 15. pii: e70203. [Epub ahead of print]41(1):
    Identification of Geographical Chemical Markers in Neolitsea pallens Using UHPLC-Q-TOF-MS/MS and Multivariate Analysis.
    Nisha Thakur, Kondeboina Murali, Yogesh Chandra Tripathi, Vinay Kumar Varshney.
       RATIONALE: Comprehensive characterization of plant metabolomes is essential for understanding phytochemical diversity and identifying location-specific metabolic signatures. However, the high complexity of untargeted metabolomics datasets requires robust analytical workflows that integrate high-resolution mass spectrometry with chemometric and pathway-based analyses. This study aimed to characterize the metabolomic landscape of Neolitsea pallens leaves collected from diverse Himalayan locations using an untargeted UHPLC-QTOF-MS/MS metabolomics approach.
    METHODS: Leaf samples collected from 13 geographically distinct Himalayan populations were analyzed using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) in negative electrospray ionization mode. Metabolites were annotated using accurate mass measurements and MS/MS fragmentation patterns. Frequency-based feature filtering was applied to refine the metabolite dataset, followed by principal component analysis (PCA) and hierarchical clustering analysis (HCA) to identify discriminant metabolites. Kyoto Encyclopedia of Genes and Genomes (KEGG)-based pathway mapping was performed to investigate the biological significance of the selected biomarkers.
    RESULTS: UHPLC-QTOF-MS/MS analysis enabled the annotation of 56 metabolites encompassing diverse primary and secondary metabolites. Frequency-based filtering combined with PCA and HCA identified 10 discriminant biomarker metabolites that effectively differentiated N. pallens populations. KEGG pathway analysis revealed that these biomarkers were predominantly associated with central carbon metabolism, with propanoate and butanoate metabolism serving as key metabolic hubs linking glycolysis, the tricarboxylic acid cycle, amino acid degradation, and fatty acid metabolism. Conserved metabolites, including citric acid, succinic acid, and β-alanine, reflected metabolic stability across populations, whereas low-frequency metabolites represented adaptive metabolic variation associated with geographical diversity.
    CONCLUSION: The integration of untargeted UHPLC-QTOF-MS/MS metabolomics, multivariate chemometric analysis, and pathway-level interpretation provides a robust strategy for resolving complex plant metabolomes and identifying biologically relevant metabolic biomarkers. This workflow offers a comprehensive framework for investigating phytochemical diversity, metabolic adaptation, and biomarker discovery in medicinal plant species.
    Keywords:  Neolitsea pallens; UHPLC–QTOF–MS/MS; chemometric analysis; propanoate and butanoate metabolism; untargeted metabolomics
    DOI:  https://doi.org/10.1002/rcm.70203
  30. PLoS One. 2026 ;21(11): e0361619
    Targeted urine metabolomics for enhanced diagnosis of hypoxic-ischemic encephalopathy in newborns: A pilot study.
    Darryl J Adamko, Maryam Alyari, Sibasis Daspal, Kim Davis, Jae Newton, Salah Almubarek, Anas El-Aneed.
      While therapeutic hypothermia (cooling treatment) is successful in preventing hypoxic ischemic encephalopathy (HIE), current diagnosis is limited to clinical features and serum lactate. Many newborns at risk of HIE are not identified within the therapeutic window and consequently miss the opportunity for cooling treatment. We hypothesized that metabolomic analysis could more accurately identify infants at risk of HIE compared to other commonly used techniques, such as serum lactate. Newborns admitted for therapeutic hypothermia for HIE, were recruited (n = 11) and compared to four healthy infants. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform was used to measure metabolites in urine samples. We identified 15 metabolites that differentiated infants with HIE from healthy controls. The metabolomic signature was subsequently utilized to blindly and accurately re-identify the same infants 3 days later, after clinical recovery, with 100% accuracy, as their metabolic profiles resembled those of non-hypoxic healthy infants. The diagnostic performance of LC-MS/MS was superior to that of lactate alone. Urine metabolomic profiling has the potential to more accurately identify neonates at risk of long-term neurologic injury than lactate alone.
    DOI:  https://doi.org/10.1371/journal.pone.0361619
  31. Drug Metab Bioanal. 2026 ;19(3): 264-272
    Sensitive Quantification of N-Nitroso Dibutylamine Impurities in Tramadol Tablets Using Optimized LC-MS/MS Method.
    G Saiteja Reddy, Nimbagal Raghavendra Naveen, M Shankar.
       BACKGROUND: NDBA, a recognized genotoxic and carcinogenic impurity in pharmaceutical products, is employed as a model compound. As a result, regulatory agencies worldwide have imposed stringent testing requirements for Nitrosamine impurities, and there is a demand for sensitive and selective analytical methods for their detection.
    OBJECTIVE: The purpose of this study was to develop and validate a Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) method for the accurate determination of NDBA impurities in Tramadol tablets, thereby overcoming the limitations of High Performance Liquid Chromatography (HPLC) in identifying low-level impurities.
    METHODS: Selectivity and sensitivity of the LC-MS/MS method were optimized, and the Limit of Quantitation (LOQ) was set to 5% and the Limit of Detection (LOD) to 3% of the specification limit. The linearity was characterised over a range of 5% to 150% relative to the specification limit. At several concentration levels within the analytical range, the precision and accuracy were determined.
    RESULTS: In the specified range, the developed method was found to be linear (R² > 0.999). Low relative standard deviation precision studies yielded results consistent with those obtained with this analytical method, and accuracy assessments demonstrated that the method could quantitatively measure NDBA over a range of concentrations. The method was suitable for routine quality control and met and exceeded regulatory performance requirements (LOQ and LOD).
    DISCUSSION: The developed LC-MS/MS method demonstrated high sensitivity, specificity, and reproducibility for the quantification of NDBA in Tramadol tablets. The method met ICH validation criteria, including acceptable precision (%RSD <5%), strong linearity (r2 = 0.9980), and satisfactory recovery (97.8-115%). The low LOD (0.002 ppm) and LOQ (0.003 ppm) confirm its suitability for detecting trace-level genotoxic impurities, ensuring regulatory compliance and pharmaceutical safety.
    CONCLUSION: It was established that a validated LC-MS/MS method provides a reliable, sensitive, and selective approach for determining NDBA impurities in Tramadol tablets. The use of this method enables compliance with the most stringent regulatory standards for pharmaceutical product safety and efficacy.
    Keywords:  LC-MS/MS; N-Nitrosodibutylamine; genotoxic impurities; impurities profiling; nitrosamines; tramadol
    DOI:  https://doi.org/10.2174/0118723128402090251024050200
  32. Anal Chem. 2026 Nov 10. 98(44): 30662-30669
    Ultrasensitive Quantification of Poloxamer 188 in Adeno-Associated Virus Products by All-Ion Fragmentation MS/MS.
    Sisi Zhang, Jing Wang, Hui Xiao, Ning Li.
      Poloxamer 188 (P188; Pluronic F68) is a nonionic surfactant frequently used in biotherapeutics, particularly in adeno-associated virus (AAV) formulations, in which it is present at trace concentrations (0.001%-0.005%). However, its accurate quantification is difficult, because of its low abundance, frequently limited sample volumes, and the ionization variability of its PEO-PPO-PEO triblock. Here, we developed an all-ion fragmentation (AIF) workflow with 44 Da and 58 Da neutral-loss-triggered data dependent (dd)-MS2 that fragments all precursors and performs quantification via the polyethylene glycol (PEG) diagnostic ion (m/z 133.1), thereby decreasing spectral complexity and adduct variability. In contrast to the most sensitive reported method, LC-MRM, using a 20 μL injection volume, which achieved a dynamic range of 250-10,000 ng/mL (0.000025%-0.001%), our method achieved a 0.00002% LLOQ with 1 μL injections, thus providing an ∼20-fold sensitivity gain that enables replicate analysis and a dynamic range 2 orders of magnitude wider (0.00002%-0.1%). Our assay is vendor-, formulation-, and LC-buffer-agnostic, and achieves ±10% accuracy and ∼10% CVs across AAV-relevant levels. Significant P188 losses attributable to container adsorption were observed; incorporation of 50% acetonitrile restored recovery and is recommended for sample preparation.
    DOI:  https://doi.org/10.1021/acs.analchem.6c03755
  33. Molecules. 2026 Oct 30. pii: 3920. [Epub ahead of print]31(21):
    A Five-Minute UHPLC-MS/MS Method Facilitates Anthocyanin Biofortification in Maize (Zea mays L.).
    Franz J O'Hanna, Shaji K Chacko, Samuel D Jesse, Alexander I Hernandez, Wenwei Xu, Timothy Paape, Michael P Dzakovich.
      Anthocyanin-rich maize offers opportunities for nutritional biofortification and natural food colorants, but conventional chromatographic methods limit the throughput needed for large-scale breeding programs. We developed a 5-min ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for quantifying maize anthocyanins and compared its performance with a 25-min method. Anthocyanins from five commercial products and one breeding line were analyzed using optimized chromatographic gradients and multiple-reaction monitoring. Anthocyanin concentration and composition varied substantially among varieties, with differing contributions from cyanidin-, peonidin-, and pelargonidin-derived compounds. The 5-min method reduced analytical runtime fivefold while preserving the relative ranking of varieties by total anthocyanin concentration, although absolute concentrations differed slightly between methods for most varieties. Random-effects modeling indicated that the methodology used to phenotype maize represented less than 1% of the total variation, further reinforcing that our rapid method is suitable for profiling breeding populations. This rapid UHPLC-MS/MS method provides a high-throughput analytical platform for quantifying predominant maize anthocyanins, facilitating phenotyping and selection for anthocyanin biofortification in maize breeding programs.
    Keywords:  anthocyanins; chromatography; cyanidin; high-throughput phenotyping; multiple-reaction monitoring; pelargonidin; peonidin; phenomics; pigmented corn; plant breeding
    DOI:  https://doi.org/10.3390/molecules31213920
  34. Anal Chem. 2026 Nov 10. 98(44): 30370-30377
    Chiral Analysis of 2-Hydroxyglutarate in IDH-mut Glioma Tissue using the Kinetic Method.
    Mahdiyeh Shahi, Jie Li, Robert Graham Cooks.
      In almost all biological systems, stereochemistry is an important aspect of the molecular structure. Aberrant molecular geometry can be a reflection of the disease state which is then manifested through effects on cellular metabolism. A well-known example occurs in isocitrate dehydrogenase (IDH)-mutant (mut) cancers, where the mutant IDH enzyme produces a unique chiral molecule, d-2-hydroxyglutarate (d-2HG). d-2HG accumulates in IDH-mut tumors and disrupts cellular pathways, including epigenetic regulation. Here, we apply the kinetic method to direct chiral analysis of 2HG in human glioma tissue. A trimeric cluster ion, [Ni(II)(Ac-l-Phe)2(l-2HG)-H]+, is formed and fragmented during tandem mass spectrometry (MS/MS). Cluster ions containing different 2HG geometry show distinctive fragment intensities. Calibration curves spanning from -100% to +100% enantiomeric excess (ee) were constructed, and the effects of potential interferences, including glutamate (Glu) and glutamine (Gln), were assessed. Using these data, we developed a chromatography-free method to determine the ee of 2HG in the glioma tissue. Chiral analysis of IDH-mut glioma tissue samples using the kinetic method was validated using LC-MS, with ee values agreeing within < 12%. Detection of predominantly d-2HG in tissue samples from (IDH)-mutant cancer is consistent with the neomorphic gain of function associated with the IDH mutation. This separation-free workflow enables rapid chiral analysis of 2HG in IDH-mut cancers and may provide complementary information to support intraoperative decision-making and precision medicine applications.
    Keywords:  brain cancer; chiral biomarker; enantiomeric analysis; molecular diagnosis; oncometabolite; tandem mass spectrometry
    DOI:  https://doi.org/10.1021/acs.analchem.6c03172
  35. Anal Chem. 2026 Nov 10. 98(44): 30906-30914
    Efficient Separation of Disaccharide Isomers Using Ion Mobility Spectrometry by Metal Cation and Anion Assisted Strategy.
    Wen Liu, Shi Chen, Xingjuan Du, Zhengwen Zhang, Jianna Yu, Guoxing Jing, Wenshan Li, Wenjie Liu.
      The rapid separation and identification of disaccharide isomers pose a significant analytical challenge in fields such as food science and glycobiology. This study developed a fast, simple, and low-cost analytical method based on electrospray ionization ion mobility spectrometry (ESI-IMS) combined with an ion adduction strategy for the efficient separation of eight common disaccharide isomers. The principle involves introducing specific metal cations or anions into the disaccharide solution to form stable adducts, thereby significantly altering their drift behavior during IMS analysis. Systematic screening of seven cations and nine anions and mass spectrometry result revealed that in positive ion mode, Cu+ forms structurally rigid dimeric adducts [Cu + 2M]+ with disaccharides, effectively amplifying subtle stereostructural differences into significant distinctions in collision cross section (CCS) and increasing the proportion of fully separated disaccharide pairs to 71.4%. In negative ion mode, I- forms predominantly single [I + M]- adducts, substantially simplifying the complex original spectra and achieving a full separation rate of 60.7%. Theoretical calculations indicated stable electronic structures within the complex systems and the differences in both the strength and interaction characteristics between the two types of adducts. This method requires no complex pretreatment or expensive advanced instrumentation, and is readily implementable on conventional drift-tube IMS platforms. It thus offers a robust, cost-effective, and high-throughput solution for disaccharide isomer analysis, with potential applications in food science, biomedicine, and materials characterization.
    Keywords:  anion adduction; disaccharide; ion mobility spectrometry; isomer separation; metal ion coordination
    DOI:  https://doi.org/10.1021/acs.analchem.6c04028
  36. Anal Chem. 2026 Nov 10. 98(44): 30161-30168
    Stable Isotope-Assisted Liquid Chromatography Mass Spectrometry Reveals Uptake, Metabolism, and Resolvin Biosynthesis from Docosapentaenoic Acid in Atlantic Salmon (Salmo Salar L.) Hepatocytes.
    Zhenxiao Zhuang, Pedro Araujo, Maren Hoff Austgulen, Bjørg Kristine Hundal.
      Docosapentaenoic acid (DPA, 22:5n-3) is an intermediate omega-3 polyunsaturated fatty acid with emerging biological significance, yet its metabolic fate in fish remains poorly defined. Stable isotope-assisted liquid chromatography-mass spectrometry (LC-MS) was applied to trace the uptake, intracellular retention, and bioconversion of deuterium-labeled DPA (DPA-d5) in primary hepatocytes from Atlantic salmon (Salmo salar L.). DPA-d5 was efficiently incorporated, and subsequent formation of both docosahexaenoic acid (DHA-d5) and eicosapentaenoic acid (EPA-d5) was observed, providing direct evidence for elongation, desaturation, and retro-conversion pathways. DPA-derived DHA was further incorporated into d-series resolvins (RvD1-d5, RvD2-d5, RvD3-d5, RvD5-d5), establishing a precursor-product relationship between DPA and bioactive lipid mediators under basal conditions. Targeted LC-MS/MS enabled unambiguous discrimination of labeled versus endogenous fatty acids. Results demonstrate the metabolic versatility of DPA in salmon hepatocytes and highlight the capacity of stable isotope-assisted LC-MS to resolve complex fatty acid pathways, including downstream mediator biosynthesis. This analytical workflow provides a powerful framework for investigating the contributions of dietary DPA, or other fatty acids, to n-3 long-chain polyunsaturated fatty acid pools and specialized pro-resolving mediators in fish.
    DOI:  https://doi.org/10.1021/acs.analchem.6c00923
  37. J Mass Spectrom Adv Clin Lab. 2026 ;39(1): 6wt6r959
    A Novel Method for the Determination of Ganciclovir Triphosphate in Human Red Blood Cells by LC-MS/MS.
    Qiao-Lan Xuan, Jun-Tao Zheng, Jia-Jia Yan, Long-Shan Liu, Ji-Feng Meng, Pan Chen, Jie Chen, Xiao Chen, Min Huang, Ke-Jing Tang, Chang-Xi Wang, Xia Zhu.
       INTRODUCTION: Ganciclovir (GCV) and its prodrug valganciclovir (VGCV) are commonly used to prevent and treat cytomegalovirus (CMV) infections in solid organ transplant recipients. Because of high interindividual pharmacokinetic variability, therapeutic drug monitoring (TDM) is important for optimizing therapy. Ganciclovir-triphosphate (GCV-TP), the active intracellular metabolite, is linked to both efficacy and toxicity; however, clinical monitoring is limited by analytical challenges.
    OBJECTIVES: To develop and validate a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying GCV-TP in human red blood cells (RBCs).
    METHODS: GCV-TP was extracted using protein precipitation, separated on a BioBasic AX column, and detected with tandem mass spectrometry using guanosine-13C10-5'-triphosphate as the internal standard.
    RESULTS: The assay was linear over 0.01-2.00 μg/mL (r 2 ≥ 0.99), with intra- and inter-assay variability ≤7.99%. GCV-TP in RBC lysate was stable when stored at room temperature for 4 h or at 4 °C for 24 h. This method was applied to RBC samples from 27 renal transplant recipients receiving VGCV, with a median GCV-TP concentration of 180.8 pmol/8 × 109 RBC (IQR: 113.6-297.6 pmol/8 × 109 RBC). GCV-TP levels showed a significant correlation with VGCV dose (p = 0.020, r = 0.31) but not with plasma GCV concentrations (p > 0.05).
    CONCLUSION: This validated method enables reliable measurement of intracellular GCV-TP and may serve as a useful tool to support individualized VGCV therapy and pharmacodynamic monitoring in clinical practice.
    Keywords:  LC-MS/MS; ganciclovir; ganciclovir-triphosphate; renal transplant recipients
    DOI:  https://doi.org/10.21627/6wt6r959
  38. J Mass Spectrom Adv Clin Lab. 2026 ;39(1): kqcara81
    Development of a Rapid and Simple LC-MS/MS Method for the Quantitation of Baricitinib in Human Plasma and Aqueous Humor.
    Xiping Li, Xian Zhang, Chengliang Zhang, Hengyi Yu, Yu Liu, Xufang Sun.
       BACKGROUND: Increasing evidence supports the off-label use of Janus kinase inhibitors for the treatment of noninfectious uveitis. Although several bioanalytical methods for determining baricitinib concentrations have been reported, no methods are currently available for monitoring concentrations at or near noninfectious uveitis lesions. This article aims to develop a rapid, sensitive, and reliable LC-MS/MS method for determining baricitinib concentrations in both plasma and aqueous humor, facilitating future clinical pharmacology research and improving therapeutic management.
    METHODS: After single-step protein precipitation and centrifugation, the samples were subjected to direct analysis. Optimal chromatographic performance was achieved using an Ultimate XB-C18 column with a mobile phase of 10 mM of ammonium formate and a mixture of methanol, acetonitrile, and isopropanol (0.2% formic acid).
    RESULTS: The authors validated the method for the limits of detection and quantification, linearity, accuracy, precision, selectivity, matrix effects, stability, and recovery, with all results meeting guidelines. This method has been successfully applied to monitor baricitinib concentrations in adult patients with noninfectious uveitis, revealing substantial variability in plasma concentrations and unsynchronized pharmacokinetic changes between plasma and aqueous humor.
    CONCLUSION: This robust method can be a valuable tool for future clinical pharmacology research and improving therapeutic management.
    Keywords:  LC-MS/MS; Noninfectious uveitis; aqueous humor; baricitinib; therapeutic drug monitoring
    DOI:  https://doi.org/10.21627/kqcara81
  39. J Mass Spectrom Adv Clin Lab. 2026 ;39(1): 3p72dt97
    Comparison of Three Urine Normalization Strategies for Mass Spectrometric Analysis in a Pediatric Asthma Model.
    Emma Finch, Maryam Alyari, Mays Al-Dulaymi, Anas El-Aneed, Darryl J Adamko.
       INTRODUCTION: Metabolomics studies the products of metabolism within a biological system to determine biomarkers for disease diagnosis. Metabolite values found in urine analysis require a method to normalize the data to account for urine concentration. Creatinine (CRN) is a commonly used method of LC-MS/MS data normalization; however, CRN is affected by muscle mass and height (HT) in children. This could be a concern when comparing children's samples across various age groups. Referencing CRN to HT (CRN/HT) is a possible strategy to correct for such a concern. Another approach is to reference each metabolite to the total concentration of the metabolites, a strategy termed the total metabolome (TM).
    METHODS: We evaluated age/HT as a confounder in pediatric metabolomic data analysis by comparing CRN, CRN/HT, or TM normalization in children aged 3-13 with or without asthma (n = 152). LC-MS/MS measured 41 metabolites in urine samples, which were normalized to the three methods. These metabolites were then used to differentiate children with asthma versus healthy children.
    RESULTS: CRN was affected by age, unlike CRN/HT or TM. The metabolites selected to differentiate healthy and asthmatic children differed based on the normalization technique; 10 metabolites were shared across models. Diagnostic accuracy was affected by the normalization strategy.
    CONCLUSION: Using CRN/HT or TM limits the variation seen across ages and was better than CRN alone. The normalization strategy for hydration affects model accuracy in pediatric research.
    Keywords:  CHILD Cohort Study; LC-MS normalization; asthma; creatinine normalization; creatinine-to-height normalization; pediatric cohort; urine metabolomics
    DOI:  https://doi.org/10.21627/3p72dt97
  40. Anal Bioanal Chem. 2026 Nov 13.
    Development of a targeted differential mobility spectrometry UHPLC-MS/MS quantitation method for free long-chain and hydroxylated fatty acids.
    Miriam Wimmer, Adam Sorbie, Olivia I Coleman, Dirk Haller, Veronika Somoza, Andreas Dunkel.
      Despite the well-established roles of long-chain fatty acids in animal and human metabolism, the involvement of their hydroxylated metabolites in carcinogenesis remains unclear, primarily due to the lack of robust quantitative analytical methods. In this study, we present a novel analytical approach that enables the quantification of cis- and trans-isomers of long-chain fatty acids and their hydroxylated metabolites in tumor and non-tumor colorectal tissues of mice. Using a combination of liquid chromatography, differential mobility spectrometry, and tandem mass spectrometry (LC-DMS-MS/MS), we achieved the separation and quantification of a diverse range of free fatty acids, including nine saturated, eleven monounsaturated, six polyunsaturated, four carboxylated, and nine hydroxylated fatty acids, covering various degrees of saturation and isomerism. The method was validated with deuterated and 13C-labelled isotopologs of non-hydroxylated fatty acids. Our comparative analysis of tumor and non-tumor colorectal tissues from mice revealed distinctive alterations in the profiles of individual analytes, particularly regarding the long-chain polyunsaturated fatty acid C20:4 and its hydroxylated metabolites C20:4-5-OH and C20:4-12-OH. These characteristic differences in concentration will enable novel insights into the potential involvement of specific long-chain hydroxylated fatty acids in the pathogenesis of colorectal tumors.
    Keywords:  Colorectal cancer; Fatty acids; Isotopic label; LC-DMS-MS/MS; Validation
    DOI:  https://doi.org/10.1007/s00216-026-06869-5
  41. Molecules. 2026 Nov 04. pii: 3971. [Epub ahead of print]31(21):
    Hybrid QuEChERS-Micro-Solid Phase Extraction for the Determination of Chloramphenicol in Honey by HPLC-MS/MS: Method Validation and Matrix-Effect Assessment.
    Tomasz Śniegocki, Elżbieta Samorek, Weronika Krajewska.
      An HPLC-MS/MS method combining modified QuEChERS-based extraction with micro-solid-phase extraction (MSPE) using C18P-PVDF microspin filters was developed and validated for chloramphenicol (CAP) determination in honey. At validation levels of 0.10, 0.15, and 0.30 µg kg-1, apparent recoveries ranged from 102.1% to 105.1%, repeatability (RSDr) from 4.5% to 9.5%, and within-laboratory reproducibility (RSDwR) from 6.0% to 12.5%. The decision limit (CCα) was 0.123 µg kg-1, below the reference point for action of 0.15 µg kg-1, while the estimated limit of detection (LOD; not experimentally verified) was 0.01 µg kg-1.. Matrix-effect assessment across 20 honey samples showed responses within the 80-120% range for the proposed procedure and the SPE and QuEChERS comparators. No CAP was detected in the authentic honey samples. The developed workflow provides a practical, validated alternative for routine CAP determination in honey.
    Keywords:  HPLC-MS/MS; QuEChERS; chloramphenicol; honey; matrix effects; micro-solid phase extraction
    DOI:  https://doi.org/10.3390/molecules31213971
  42. Forensic Sci Int. 2026 Nov 11. pii: S0379-0738(26)00379-8. [Epub ahead of print]391 113191
    Forensic toxicological application of a targeted UPLC-MS/MS method for 15 controlled and prescription-related drugs in human hair: Validation and analysis of 369 anonymized authentic samples.
    Xiao Wang, Haoyue Zhang, Bin Di, Mengxiang Su.
      Targeted hair analysis is an important approach for the retrospective assessment of drug exposure in forensic toxicology. In this study, a sensitive and practical UPLC-MS/MS method was developed and validated for the simultaneous determination of 15 controlled and prescription-related drugs in human hair, including buprenorphine, triazolam, ketamine, diazepam, nitrazepam, clonazepam, oxazepam, lorazepam, estazolam, midazolam, chlordiazepoxide, tramadol, pethidine, cocaine, and diphenoxylate. Hair samples were prepared by high-speed milling followed by ultrasonic extraction with methanol, and the analytes were detected by multiple reaction monitoring in positive electrospray ionization mode. The method showed good linearity over the concentration range of 0.005-2.5 ng/mg, with correlation coefficients of r ≥ 0.99 for all analytes. The lower limit of quantification was 0.005 ng/mg for each analyte. Intra-day and inter-day precision were below 15%, accuracy ranged from 85% to 115%, and absolute recovery ranged from 88% to 106%. The validated method was further applied to 369 anonymized authentic hair samples collected from selected populations. Tramadol was the most frequently detected analyte, with 36 positive cases and concentrations ranging from 0.01 to 222 ng/mg. Buprenorphine, diazepam, and midazolam were each detected in one sample. Overall, the proposed method provides a practical targeted tool for routine hair testing of selected controlled and prescription-related drugs in forensic toxicology laboratories.
    Keywords:  Controlled drugs; Forensic toxicology; Hair analysis; Tramadol; UPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.forsciint.2026.113191
  43. J Agric Food Chem. 2026 Nov 11. 74(44): 34885-34899
    Spatial Metabolomics Reveals Tissue Specificity and Distribution of Metabolites in Paeonia lactiflora Roots.
    Jin Lu, Junyi Wang, Chengda Zhang, Yang Liu, Jia Hao, Xiaoyan Wang, Yanxu Chang.
      Various secondary metabolites accumulate in the roots of Paeonia lactiflora Pall. (P. lactiflora) and exhibit therapeutic potential for multiple disorders. However, their enrichment sites in different tissues or parts of the roots remain unclear. In this study, metabolites in different parts of P. lactiflora roots were imaged by air-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) to acquire full-scan MS1 data. They were classified by a mass defect filtering (MDF) model into two precursor ion lists (PILs), which guided metabolite identification using ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS). Ultimately, 92 metabolites were identified, and distributions of monoterpenoids and tannins were revealed. From the root-tip segment (S) to the rhizome-proximal segment (L), metabolites exhibited a spatial transition from diffuse to annular distribution, with enhanced tissue specificity. Furthermore, 19 differential metabolites were screened and characterized. This study facilitates sustainable utilization of P. lactiflora resources and offers insights for plant metabolomics.
    Keywords:  AFADESI-MSI; P. lactiflora; UHPLC-Q-TOF-MS/MS; different tissues or parts of roots; spatial metabolomics
    DOI:  https://doi.org/10.1021/acs.jafc.6c10133
  44. Int J Mol Sci. 2026 Nov 08. pii: 10012. [Epub ahead of print]27(21):
    AI-Assisted Molecular Structure Prediction from Tandem Mass Spectra: Reliability and Validation.
    Jun Sang Yu, Sung Hue Jeong, Yejin Kim, Dong Keun Lee, Hye Hyun Yoo.
      Artificial intelligence (AI) has broadened the interpretation of tandem mass spectra (MS/MS) beyond experimental library matching, enabling molecular fingerprint prediction, candidate ranking, and generation of previously unlisted structures. Yet these outputs do not all support the same claim about chemical identity. This review briefly summarizes database-restricted retrieval, database-free generation, and scaffold-guided hybrid methods, and then asks what additional evidence is needed to turn a plausible structure into a defensible annotation. We examine failure modes involving incomplete candidate spaces, erroneous precursor assignments, structurally related isomers, leakage between training and test data, and misinterpreted confidence scores. The review contributes two practical elements: a candidate-discrimination workflow, in which alternative structures are retained and targeted measurements such as multienergy fragmentation, retention behavior, ion mobility, and authentic standards are selected according to the unresolved structural question; and an evidence-to-claim checklist that links each question to the most informative measurement and to the strongest supportable identification confidence level. Both synthesize established analytical principles for small-molecule electrospray LC-HRMS/MS rather than constituting a validated scoring algorithm. By making the basis of each annotation transparent, they are intended to improve the practical value of AI-assisted MS/MS in metabolomics, pharmaceutical analysis, environmental screening, and forensic toxicology.
    Keywords:  artificial intelligence; database retrieval; molecular generation; structure prediction; tandem mass spectrometry
    DOI:  https://doi.org/10.3390/ijms272110012
  45. Biomed Chromatogr. 2026 Dec;pii: e70656. [Epub ahead of print]40(12):
    Quantitative Analysis of Branaplam (LMI070) in Human Plasma by LC-MS/MS in Support of Clinical Trial in Infant Patients With Spinal Muscular Atrophy (SMA).
    Yunlin Fu, Wenkui Li, Franck Picard.
      When in vivo bridging between conventional venous sampling and patient centric capillary sampling (or microsampling) is not feasible for pharmacokinetic (PK) assessment, collection of small volumes of venous plasma is a viable approach to support regulated clinical trial in pediatric populations. A sensitive LC-MS/MS method was developed and validated for the quantification of LMI070, a drug candidate for the treatment of spinal muscular atrophy (SMA), in plasma samples collected from a first-in-human, first-in-patient trial in infant patients. The method has a dynamic range of 0.200-200 ng/mL using 20-μL sample volume. The accuracy and precision of the method along with other validation parameters met the predefined requirements. The method was successfully implemented in timely analysis of plasma samples to support PK/PD and other assessments of the trial.
    Keywords:  LC‐MS/MS; branaplam (LMI070); clinical sample analysis; pediatric trials; spinal muscular atrophy (SMA)
    DOI:  https://doi.org/10.1002/bmc.70656
  46. Metabolomics. 2026 Nov 13. pii: 175. [Epub ahead of print]22(6):
    Metabolomics and lipidomics in the differentiation of CIN 2 and CIN 3: an exploratory pilot study.
    Luana Quadros de Souza Leão, Giovanna Melo Marques, Adriano de Souza Carolino, Ana Carolina Rosa da Silva, Sofia Angiole Cavalcante, Elzalina Ribeiro Soares, Jonas Balan de Padua, Anderson Ferreira Gonçalves, Marina Amaral Alves, Rafael Garrett, Priscila Ferreira de Aquino.
       INTRODUCTION: High-grade cervical intraepithelial neoplasia (CIN 2-3) is a precursor lesion to cervical cancer and, although it presents different probabilities of regression and progression, it is usually grouped together in screening and managed similarly. However, the mechanisms that determine its evolution are still poorly understood, and standard treatment by conization can lead to reproductive complications such as subfertility and premature birth.
    OBJECTIVE: This study aimed to identify metabolic and lipid signatures that can distinguish between CIN 2 and CIN 3 lesions using liquid chromatography-high-resolution mass spectrometry (LC-HRMS).
    METHODS: Tissue samples from the lesion and adjacent margin of 24 women with CIN 2-3 were subjected to metabolomic and lipidomic analysis by LC-HRMS. Multivariate statistical analyses, pathway analysis, and correlation were performed to understand these neoplasms' development and progression and identify potential metabolites and lipids to distinguish such lesions.
    RESULTS: Metabolomic and lipidomic analysis by LC-HRMS in cervical tissue identified molecular signatures with differential abundance between CIN 2 and CIN 3, highlighting patterns associated with the degree of lesion. Multivariate analyses highlighted metabolites (serine, glutamic acid, guanidine-6-monophosphate, succinic acid, mannose 6-phosphate, and taurine) and lipids (TG 49:1, TG 50:1, and TG 52:2) as the main contributors to the discrimination between the groups, suggesting relevant biological alterations in high-grade cervical lesions.
    CONCLUSION: Combination metabolomic and lipidomic approach enabled the identification of metabolites and lipids potentially involved in the pathogenesis of intraepithelial neoplasia (CINs). In addition, these identified metabolite and lipid panels are promising as complementary tools for future screening strategies for these lesions.
    Keywords:  Cervical intraepithelial neoplasia; LC-HRMS; Lipidomics; Metabolomics
    DOI:  https://doi.org/10.1007/s11306-026-02555-w
This page is being maintained by Thomas Krichel. It was last updated on 2026‒11‒15 at 0:40.