bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2026–05–31
39 papers selected by
Sofia Costa, Matterworks



  1. Molecules. 2026 May 16. pii: 1684. [Epub ahead of print]31(10):
      Following the passage of the Agriculture Improvement Act of 2018, demand for accurate cannabinoid quantification in hemp flowers has increased to ensure regulatory compliance. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a triple-quadrupole mass spectrometer provides high sensitivity and selectivity and is well suited for this purpose; however, a review of the literature indicates that many published LC-MS/MS methods target only a limited number of cannabinoids, and reliable differentiation of structural isomers remains challenging. In this study, an LC-MS/MS method was developed for the simultaneous quantification of eighteen cannabinoids in hemp flowers. Baseline chromatographic separation of structural isomers enabled reliable differentiation of compounds with highly similar fragmentation patterns and allowed the use of the most sensitive multiple reaction monitoring (MRM) transitions for quantification. Both positive and negative ionization modes were employed to achieve optimal sensitivity using dynamic polarity switching within a single analytical run. Following validation in accordance with ISO/IEC 17025, the method was applied to a proficiency test hemp sample and six commercial hemp samples, demonstrating excellent time efficiency (11 min for 18 cannabinoids) and an exceptionally wide calibration range (8-5000 ng/mL, corresponding to 0.032-20% (w/w) for all cannabinoids).
    Keywords:  MRM; cannabinoids; hemp flowers; liquid chromatography; quantification; triple-quadrupole mass spectrometer
    DOI:  https://doi.org/10.3390/molecules31101684
  2. J Proteome Res. 2026 May 27.
      Liquid chromatography-mass spectrometry is a potent and robust tool for studying metabolism. However, conventional workflows can suffer from poor peak shapes, limited pressure tolerance, coelution of polar metabolites, and unstable retention times. Here, we describe the development of a more stable HILIC method for LC-MS metabolomics of human plasma and cell extracts, optimizing a zwitterionic HILIC (Z-HILIC) column for improved untargeted performance. We found that using high-pH ammonium bicarbonate with 90% acetonitrile in mobile phase B (ABC B) can greatly improve peak shapes of select metabolites when compared to 100% acetonitrile (ACN B), but at the cost of poor retention time stability. We therefore focused on optimizing chromatography for the ACN B method and observed that cooling the column to 5 °C substantially enhanced peak shape for the BEH-bound Z-HILIC and amide columns but had little effect on the polymeric ZIC-pHILIC column. The low-temperature method with the Z-HILIC column (LT-ZHILIC) enables high-resolution separation of 471 metabolite library standards and from both cellular extracts and human plasma and demonstrates robust stability over 100 consecutive injections and multiple days. Application of the untargeted LT-ZHILIC method to characterize the metabolic consequences of glutamine and pyruvate deficiency in human cells revealed a striking change in nucleotide phosphates─a perturbation that was not observed in the ZIC-pHILIC analysis of the same samples likely due to inadequate elution profiles. In sum, the LT-ZHILIC workflow offers a robust platform to advance untargeted metabolomics by improving metabolite coverage, resolution, and retention time stability, making it a promising technique for providing novel insights into cellular metabolic rewiring and the human plasma metabolome.
    Keywords:  HILIC; metabolomics; nucleotides; separation
    DOI:  https://doi.org/10.1021/acs.jproteome.5c01216
  3. Rapid Commun Mass Spectrom. 2026 Aug 30. 40(16): e70116
       INTRODUCTION: Erdafitinib is a pan-FGFR tyrosine inhibitor approved for urothelial carcinoma, with growing interest in its broader anticancer applications. Accurate quantification in preclinical models is essential for understanding pharmacokinetics and optimizing therapeutic use. Developing a sensitive, economical bioanalytical method ensures reliable drug monitoring, supporting translational research and regulatory compliance in oncology drug development.
    METHODS: Rat plasma samples were processed by protein precipitation, and analytes were separated on a reverse-phase column using a gradient mobile phase. Detection employed LC-MS/MS in positive ionization mode with multiple reaction monitoring (MRM), monitoring m/z 447.1 → 362.1 for erdafitinib and m/z 515.2 → 276.0 for internal standard. The total run time was 2.0 min.
    RESULTS: The method demonstrated excellent linearity across 1.0-1000 ng/mL with a lower limit of quantification of 1.0 ng/mL. Precision was < 12% and accuracy within 100% ± 6% across the calibration range. The assay was validated according to US FDA M10 guidelines and successfully applied to pharmacokinetic studies in Sprague Dawley rats, enabling reliable plasma concentration profiling of erdafitinib.
    CONCLUSIONS: This validated LC-MS/MS method provides a rapid, sensitive, and cost-effective approach for erdafitinib quantification in preclinical plasma samples. Its application to pharmacokinetic studies supports drug development by enabling accurate assessment of exposure and disposition, thereby advancing pharmacological understanding and facilitating translation to clinical oncology research.
    Keywords:  FGFR; LC‐MS/MS; bioanalysis; erdafitinib; telmisartan; tyrosine kinase inhibitor
    DOI:  https://doi.org/10.1002/rcm.70116
  4. Anal Bioanal Chem. 2026 May 27.
      Spatially resolved metabolomics based on mass spectrometry imaging (MSI) enables in situ characterization of tissue-specific metabolic functions by mapping the spatial distribution of metabolites. However, accurate metabolite annotation and automated analysis of large-scale MSI data remain challenging, mainly due to mass-to-charge ratio (m/z) shifts and dependence on generic databases. To address these challenges, we developed the MSI Data Analysis Tool (MSIDAT), an automated and user-friendly MSI data processing platform. By integrating liquid chromatography-tandem mass spectrometry (LC-MS/MS)-assisted metabolite identification, customized metabolite ion databases can be constructed to improve the specificity and reliability of metabolite annotation. In addition, m/z shifts in MSI data were systematically evaluated using endogenous reference ions by calculating the relative mass error between theoretical and measured m/z values, enabling adaptive mass tolerance correction. Based on this strategy, mass error-informed metabolite matching and putative annotation were achieved. Furthermore, MSIDAT provides flexible parameter settings, modular workflows, and open-source accessibility, facilitating efficient and reproducible MSI data analysis. The performance of the platform was demonstrated in a clinical cohort of rectal cancer patients, in which hundreds of metabolites were putatively annotated and spatial alterations in tumor-associated metabolites were observed, suggesting fatty acid-related metabolic alterations. Overall, this study presents a robust and versatile analytical platform for improving metabolite annotation in MSI, thereby enhancing data mining efficiency and supporting spatial metabolomics-driven biomarker discovery and clinical applications.
    Keywords:   m/z shift evaluation; Data processing tool; Mass spectrometry imaging; Metabolite database; Spatially resolved metabolomics
    DOI:  https://doi.org/10.1007/s00216-026-06576-1
  5. Anal Chim Acta. 2026 Aug 01. pii: S0003-2670(26)00510-6. [Epub ahead of print]1409 345560
       BACKGROUND: The diversity of ergot alkaloids (EAs) isomers poses significant challenges for traditional analytical methodologies. By adding an orthogonal rapid gas-phase separation step, Ion mobility mass spectrometry (IM-MS) enables faster workflows and higher throughput without sacrificing data quality. However, its application in this context remains limited. We hypothesize that high resolution IM-MS can shorten chromatographic analysis times for natural compounds while maintaining or even enhancing separation performance.
    RESULTS: In this study, we developed a high-throughput LC-cyclic IMS (cIMS) methodology for the analysis of 28 EAs including 9 isomeric pairs, which comprises epimers, conformational isomers and constitutional isomers, within 4 min by leveraging the additional dimension of separation provided by the cyclic ion mobility with high-resolution mass spectrometry. A 60% reduction in analysis time has been shown using LC-cIM-MS compared to LC-MS while keeping selectivity of the method. Ion mobility was particularly effective for the separation of small (<300 Da) and more polar conformational and constitutional isomers with peak-to-peak resolution, outperforming chromatographic separation.
    SIGNIFICANCE: These results highlight the importance of complementary separation dimensions for the analysis of complex mixtures of natural compounds. The method developed in study allows to separate currently regulated EAs epimers within 4-min LC-cIM-MS run.
    Keywords:  CCS; Ergot alkaloids; Food safety; cyclic ion mobility
    DOI:  https://doi.org/10.1016/j.aca.2026.345560
  6. Brief Bioinform. 2026 May 04. pii: bbag247. [Epub ahead of print]27(3):
      Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy are complementary analytical techniques widely used in proteomics, metabolomics, and structural biology. Both generate high-dimensional, noisy spectra where overlapping peaks complicate interpretation. LC-MS relies on retention time (RT) separation before mass analysis, while multidimensional NMR spreads information across chemical-shift axes to reduce congestion. However, comparative or replicate experiments often introduce RT shifts in LC-MS or frequency shifts in NMR, hindering accurate matching of corresponding features. In some experiments, such as variable-temperature NMR, the shifts are intentionally triggered, and frequency tracking provides important information. In any case, a robust, scalable alignment across runs is critical for reliable compound identification, quantification, and structural analysis. We propose a truncated Wasserstein distance-based algorithm for aligning LC-MS and NMR spectra. By constraining maximum transport distance and formulating alignment as a minimum-cost flow problem solved via the Network Simplex algorithm, our method accelerates computation, suppresses spurious matches, and improves robustness to noise. On benchmark LC-MS datasets, it achieved 0.97 precision, 0.96 recall, and a 0.6-s runtime, outperforming OpenMS and DeepRTAlign tools. For NMR data, the algorithm proved effective in 2D, 4D, and even 7D analyses. The algorithm is implemented in wnetalign with supporting modules wnet and pylmcf, available on PyPI and GitHub under permissive licenses: https://github.com/michalsta/pylmcf, https://github.com/michalsta/wnet, https://github.com/michalsta/wnetalign.
    Keywords:  Wasserstein distance; liquid chromatography–mass spectrometry; network simplex algorithm; nuclear magnetic resonance spectroscopy; optimal transport; spectra alignment
    DOI:  https://doi.org/10.1093/bib/bbag247
  7. bioRxiv. 2026 May 11. pii: 2026.05.06.723373. [Epub ahead of print]
      Tandem mass spectrometry (MS/MS) fragments molecules into smaller pieces, generating spectra composed of m/z values and intensities that encode structural information for molecular annotation. With increasing mass spectrometry data acquisition speeds, manual annotation from MS/MS lags far behind data generation and remains a bottleneck in metabolite annotation. Current computational methods, such as molecular networking, address this challenge by organizing similar structures into families of related compounds. However, they generally provide only similarity scores, offering weak actionable insights for structural annotation. To address this limitation, we present the Molecular Transformation Graph Edit Measure (MT-GEM), a distance metric that quantifies discrete structural transformations between molecules through graph edge removals that approximate structural modifications. Building on this metric, we developed an ensemble machine learning architecture, the Spectrum Transformation Edit Predictor (STEP), that builds upon TransExION and DREAMS to predict MT-GEM distances from MS/MS spectra. STEP achieves an average precision of 48.4% for identifying single structural transformations between MS/MS pairs, representing more than a tenfold improvement over state-of-the-art similarity metrics, including spectral entropy similarity (3.8%) and modified cosine (2.5%). On experimental human gut microbial community data, STEP identifies 3 times more single-transformation metabolite pairs than feature-based molecular networking at equivalent precision. In a discovery application, STEP highlights one drug metabolite and two new natural product analogs missed by modified cosine in feature-based molecular networking. By providing discrete transformation predictions rather than continuous similarity scores, MT-GEM and STEP enable hypothesis-driven metabolite annotation with testable structural modifications, which we envision will accelerate discovery of new molecules from MS/MS metabolomics datasets.
    DOI:  https://doi.org/10.64898/2026.05.06.723373
  8. Biomed Chromatogr. 2026 Jul;40(7): e70498
      ARV-393 is a novel PROTAC designed to degrade B-cell lymphoma 6. To support its further development, it is necessary to disclose the pharmacokinetic and metabolism profiles. To achieve this goal, in this study, a simple and sensitive UPLC-MS/MS method was developed for the quantification of ARV-393 and a UPLC-Q-Exactive Orbitrap-HRMS method was utilized to identify its metabolites in dog plasma. After acetonitrile-mediated protein precipitation, the sample was separated on a Waters ACQUITY BEH C18 column (50 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of 2 mM of ammonium acetate and acetonitrile, each containing 0.1% formic acid, delivered at 0.4 mL/min. The analyte and internal standard were quantified using the transitions of m/z 898.2 → 398.2 and m/z 724.4 → 396.2, respectively. The method demonstrated excellent linearity over the concentration range of 1.0-1000 ng/mL (r > 0.998). The validated method was successfully applied to a pharmacokinetic study of ARV-393 in dogs. The results indicated that ARV-393 exhibits low clearance and favorable bioavailability (64.7%). Four metabolites were tentatively identified by accurate mass and fragment ions. The proposed metabolic pathways include hydroxylation and hydrolysis. This work provides an overview of the pharmacokinetic and metabolism of ARV-393, laying a foundation for further development.
    Keywords:  ARV‐393; BCL6 PROTAC; metabolite; method validation; pharmacokinetics
    DOI:  https://doi.org/10.1002/bmc.70498
  9. Toxins (Basel). 2026 May 06. pii: 219. [Epub ahead of print]18(5):
      Mycotoxin contamination in wheat products has consistently been a key issue of concern in food safety, and urinary biomonitoring provides an effective approach for assessing internal human exposure. In this study, a sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of 28 mycotoxins in wheat products and human urine. For the two matrices, the extraction solvent, acid concentration, solid-phase extraction cartridge type, and enzymatic hydrolysis parameters were optimized. Under the optimized conditions, all target compounds showed excellent linear relationships within the tested concentration ranges (R2 > 0.99). In wheat products, the spiked recoveries ranged from 70.2% to 120%, the repeatabilities ranged from 1.6% to 9.1%, and the limits of detection and limits of quantification were 0.001~8.3 μg/kg and 0.002~25.0 μg/kg, respectively. In urine, the spiked recoveries ranged from 79.3% to 120%, the repeatabilities ranged from 0.7% to 9.4%, and the limits of detection and limits of quantification were 0.0001~1.0 μg/L and 0.0002~3.0 μg/L, respectively. Analysis of real samples showed that at least seven mycotoxins were detected in wheat product samples, and at least five were detected in urine samples. In wheat products, the detection rates of deoxynivalenol, enniatin B, enniatin A1, enniatin B1, tenuazonic acid, and tentoxin were all 100%, whereas in urine, the detection rate of fumonisin B1 reached 100%, and tenuazonic acid showed the highest mean concentration in both matrices. In conclusion, the developed ultra-performance liquid chromatography-tandem mass spectrometry method is suitable for the simultaneous quantification of 28 mycotoxins in wheat products and human urine, and its preliminary application demonstrates good practical applicability.
    Keywords:  UPLC-MS/MS; human urine; mycotoxins; wheat products
    DOI:  https://doi.org/10.3390/toxins18050219
  10. Metabolites. 2026 May 20. pii: 344. [Epub ahead of print]16(5):
      Background: Coenzyme Q10 and Alpha-lipoic acid are two essential antioxidants involved in numerous physiological processes, including cellular energy production and the mitigation of oxidative stress. Their accurate quantification is critical for understanding their biological roles and therapeutic potential. Herein, an RPLC-MS/MS method for the rapid and simultaneous determination of ubiquinone-10 (CoQ10), the reduced form ubiquinol-10 (CoQ10H2), and Alpha-lipoic acid (ALA) in human serum was developed and validated. Methods: Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 150 mm, i.d. 1.7 μm). Detection was performed on a SCIEX Triple Quad 6500+ system, applying multiple reaction monitoring (MRM). Single-phase protein precipitation was selected as the sample preparation protocol, providing satisfactory recovery for the analytes. Results: The method was linear over the concentration of 53.8-613 ng/mL for CoQ10H2, 23.1-263 ng/mL for CoQ10 and 7.7-87.6 ng/mL for ALA. Intra- and inter-day accuracy was found to be between 81.8 and 109% and 84.4 to 106%, respectively, for all analytes, while intra- and inter-day precision was found to vary from 0.8% to 9.9% %RSD and 2.0% to 7.7% %RSD, respectively. A limit of quantitation (LOQ) of 4.2 ng/mL was found for CoQ10H2, 1.7 ng/mL for CoQ10 and 0.7 ng/mL for ALA. Conclusions: The developed LC-MS/MS method enables rapid, sensitive and simultaneous quantification of CoQ10H2, CoQ10, and ALA in human serum with satisfactory accuracy, precision and sensitivity. The method is suitable for bioanalytical applications and was successfully applied to the analysis of 10 real samples obtained from healthy volunteers.
    Keywords:  Alpha-lipoic acid; coenzyme Q10; method development; oxidative stress
    DOI:  https://doi.org/10.3390/metabo16050344
  11. Pharmaceuticals (Basel). 2026 May 19. pii: 795. [Epub ahead of print]19(5):
      Background: Abemaciclib (ABM) in combination with tamoxifen (TAM) is an extremely significant treatment regimen for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer. It is approved for patients to reduce the risk of cancer recurrence. A bioanalytical method for the simultaneous determination of this new anti-breast cancer combination and its pharmacokinetic application has not yet been reported. Methods: An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying ABM, TAM, and its metabolites, including abemaciclib active metabolites M2, M18, and M20 and tamoxifen active metabolite N-desmethyl tamoxifen (NDTAM), in rat plasma using econazole as the internal standard (IS). Chromatographic separation was achieved on a Kinetex C18 column (100 × 2.1 mm ID, 2.6 µm) using gradient elution with 5 mM ammonium formate in water (eluent A) and 5 mM ammonium formate in water/methanol (1:9, v/v, eluent B) at a flow rate of 0.4 mL/min. Detection was performed on a TSQ Fortis Plus mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. Results: The developed method was validated according to the guidance of the FDA. Linearity in rat plasma (ng/mL) was achieved from 1 to 1000 for ABM, TAM, and M20; 3 to 1000 for M2; 5 to 500 for M18; and 1 to 500 for NDTAM; with correlation coefficients ranging from 0.9991 to 0.9931 for all analytes using a weighting factor of 1/X2. The lower limit of detection (LLOD) ranged between 0.3 and 1.5 ng/mL for all drugs. The accuracy ranged from 96 to 108% and the precision was less than 7.6% RSD for all analytes. For the first time, the newly developed approach was effectively used in a pharmacokinetic study on the simultaneous oral administration of ABM and TAM in rats that received 30.0 mg/kg of ABM and 8.0 mg/kg of TAM. Conclusions: To the best of our knowledge, this is the first reported UPLC-MS/MS method for the assay of ABM, TAM, and its active metabolites in plasma. This method offers a bioanalytical tool for assessing the pharmacokinetics of ABM and TAM. Therefore, this study makes a definite significant contribution to the field of bioanalytical research. Further validation in human plasma is required for future clinical or therapeutic drug monitoring applications, as the approach was developed in an animal model.
    Keywords:  UPLC–MS/MS; abemaciclib; breast cancer; metabolites; pharmacokinetics; rat plasma; tamoxifen
    DOI:  https://doi.org/10.3390/ph19050795
  12. Methods Enzymol. 2026 ;pii: S0076-6879(26)00075-3. [Epub ahead of print]730 169-176
      Natural products (NPs) are valuable sources for drug development, as they are often associated with potent bioactivities. Therefore, approaches that enable the rapid discovery of novel NPs by mining their presence in crude extracts metabolomics data while providing insights into their novelty are highly sought after by chemists. Mass spectrometry-based NPs targeted discovery often relies on a complicated decision-making process involving tedious comparison of exact masses data and tandem mass spectra-based annotation tools output against various databases. To address this bottleneck, we developed MS2DECIDE, for which a tutorial workflow for the targeted discovery of novel NPs is outlined in this chapter.
    Keywords:  Decision-making; Dereplication; Mass spectrometry; Multiannotation; Natural products; Prioritization
    DOI:  https://doi.org/10.1016/bs.mie.2026.03.002
  13. Metabolomics. 2026 May 29. pii: 89. [Epub ahead of print]22(3):
       INTRODUCTION: Blood microsampling (BµS) devices collect less than 100 µL of blood, offering a less invasive and more cost-effective alternative to venipuncture. However, its metabolomic comparability to conventional samples remains unclear, and standardized BµS metabolomic workflows are lacking.
    OBJECTIVES: This study evaluated the impact of using three BµS devices (Mitra®, Capitainer®, and Whatman™ 903) on the metabolomic interpretation of human biomonitoring samples. We compared them to conventional samples (plasma and whole blood) and evaluated the interplay of different analytical conditions.
    METHODS: Venous blood from 10 adults (5 males, 5 females) was sampled onto the three devices. First, three agitation conditions (ultrasound, shaker, and homogenizer) were evaluated at three blood concentrations (1.5%, 5.5%, and 11%). The optimized method was then used to compare the metabolite profiles between BµS devices, whole blood, and plasma. Reverse-phase and hydrophilic-interaction chromatography, in positive and negative ionization modes, were combined for liquid chromatography-mass spectrometry (LC-MS) analysis.
    RESULTS: All agitation conditions and concentrations proved suitable for BµS untargeted metabolomics. Combining different analytical modes and fragmentation ranges proved helpful for maximizing metabolite coverage. BµS-derived metabolite profiles aligned more closely with whole blood than plasma. Some metabolites were more characteristic of a sample type, whereas others were common across sample types. All sample types enabled sex-based differentiation, with metabolites such as amino acids, lipids, and acylcarnitines driving the separation.
    CONCLUSIONS: These findings enhance our understanding of BµS metabolite coverage and highlight its potential in human biomonitoring. The choice of device depends on the application and the metabolites of interest, offering flexibility for clinical use and research.
    Keywords:  Blood microsampling (BµS); Capitainer® ; Dried blood spots (DBS); Liquid chromatography–mass spectrometry (LC-MS); Metabolomics; Volumetric absorptive microsampling (VAMS)
    DOI:  https://doi.org/10.1007/s11306-026-02424-6
  14. J Pharm Biomed Anal. 2026 May 19. pii: S0731-7085(26)00243-8. [Epub ahead of print]279 117575
      Non-stimulant medications, such as atomoxetine (ATX) and guanfacine (GFC), are increasingly prescribed for attention-deficit/hyperactivity disorder (ADHD), including among women of childbearing age. However, experimental data on the transfer of these drugs into human breast milk remain extremely limited. In this study, we developed and validated a highly sensitive and precise ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of ATX, its major metabolites (4-hydroxy ATX: 4-OH ATX and N-desmethyl ATX: N-DAT), and GFC in human plasma and breast milk. Stable isotope-labeled compounds were used as internal standards (IS). Plasma samples were prepared using liquid-liquid extraction with ethyl acetate, and breast milk samples were treated by protein precipitation with acetonitrile containing 0.5% formic acid. The method required only 100 µL of sample volume and showed good linearity, accuracy, and precision over the tested ranges (plasma: 0.5-50 ng/mL; breast milk: 0.8-50 ng/mL). Matrix effects were evaluated using six individual lots of plasma and breast milk, and acceptable variability was achieved. The validated method was applied to plasma and breast milk samples from an ATX-treated lactating woman, yielding milk-to-plasma ratios (M/P) of 0.089 for ATX and 1.17 for 4-OH ATX. The relative infant dose (RID) was estimated at 0.086% for ATX and 0.059% for 4-OH ATX, and the combined RID was estimated to be 0.145%. This study provides a robust and reliable tool for assessing the transfer of non-stimulant ADHD medications into human breast milk and strengthens the evidence base for safe pharmacotherapy during lactation.
    Keywords:  Atomoxetine; Breast milk; Guanfacine; UPLC–MS/MS
    DOI:  https://doi.org/10.1016/j.jpba.2026.117575
  15. Anal Chem. 2026 May 26.
      Metabolomics has emerged as a mainstream approach for investigating the complex metabolic underpinnings of living systems, and over recent years, it has increasingly been applied to large cohort studies that tax the limits of existing computational tools. Most existing metabolomics software tools are effective at analyzing small data sets but exhibit a number of shortcomings that limit their utility when applied to large studies: they store entire data sets in memory, they use batch-dependent fitting algorithms, and they do not use concrete metrics for peak fitting, which not only results in inconsistent peak-picking results across samples but also complicates the documentation of data analyses. To address this, we developed the mass-spectrometry metabolomics integrator (MS-MINT), a Python application for processing, analyzing, and visualizing large liquid chromatography-mass spectrometry (LC-MS) data sets. To enable reproducible large-scale data processing, MS-MINT uses a region of interest (ROI)-based approach to extract data. We illustrate the function of this new tool by analyzing metabolites present in the media of a large data set (3334 files) of Staphylococcus aureus cultures. We show that MS-MINT accurately reproduces data generated from other software tools in a fraction of the time. In summary, MS-MINT offers a purpose-built software platform to support large-scale metabolomics data analyses. MS-MINT software is freely available at https://www.lewisresearchgroup.org/software.
    DOI:  https://doi.org/10.1021/acs.analchem.6c01083
  16. J Xenobiot. 2026 May 03. pii: 77. [Epub ahead of print]16(3):
      Toxicological testing for drugs of abuse (DOAs) is an essential tool for healthcare practitioners and law enforcement agencies. Oral fluid (OF) is an alternative biological fluid for detecting recent DOA intake and is widely employed in forensic investigations. In the current study, a relatively novel and "green" fabric phase sorptive extraction (FPSE) procedure for sample preparation was coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to provide simplicity, cost-effectiveness, rapidity, low solvent consumption, and high analytical performance for the quantitative determination of ten commonly encountered DOAs and metabolites: amphetamine, benzoylecgonine, cocaine, codeine, ecgonine methyl ester, methadone, methamphetamine, 3,4-methylenedioxyamphetamine, 6-monoacetylmorphine, and morphine. The FPSE procedure was optimized by testing different filters, pH, extraction time, and solvents. The validated method demonstrated excellent linearity for all analytes, selectivity, acceptable precision, and high sensitivity (ranges for limits of detection (LODs) and quantification (LOQs) were 0.01-2 ng/mL and 0.03-6 ng/mL, respectively). Autosampler and short-term freeze stability exceeded 95% and 90% for all analytes, respectively. Overall, the combination of FPSE with LC-MS/MS provided a sensitive, selective, and environmentally friendly innovative analytical approach for the determination of DOA in OF and is suitable for both screening and confirmatory forensic and clinical applications.
    Keywords:  LC-MS/MS; drugs of abuse; fabric phase sorptive extraction; forensic toxicology; oral fluid; validation
    DOI:  https://doi.org/10.3390/jox16030077
  17. Talanta. 2026 May 22. pii: S0039-9140(26)00684-3. [Epub ahead of print]309 130028
      We report a chemometrically optimized high-throughput analytical workflow integrating filtration-assisted dispersive micro-solid phase extraction (FA-D-μSPE) with LC-MS/MS for quantitative profiling of ten major human bile acid-3-sulfates (BA-S) in urine. BA-S are important urinary metabolites of bile acid detoxification and promising non-invasive biomarkers of hepatobiliary disorders. The proposed workflow implements dispersive μSPE in a 96-well filtration format, enabling in-plate sorbent extraction without centrifugation, magnetic separation, or manual phase separation. Method development was guided by sequential experimental designs (Plackett-Burman, central composite, and Box-Behnken), allowing statistically guided optimization of chromatographic separation, mass spectrometer settings, and extraction conditions. A custom 3D-printed dispenser enabled fast and reproducible in-house loading of extraction sorbent into filtration plates and facilitated flexible customization of sorbent chemistry. Compared with conventional cartridge-based or well plate SPE, FA-D-μSPE reduces solvent consumption, processing time, and consumable costs while maintaining robust analytical performance. Due to the endogenous presence of BA-S in human urine, method validation was performed using rat urine as a surrogate matrix. The method was validated according to ICH M10 guidelines, demonstrating linearity over 10-4000 nmol L-1, high accuracy and precision, and controlled matrix effects. The proposed FA-D-μSPE LC-MS/MS method provides a robust and cost-efficient strategy for rapid quantitative bioanalysis of urinary BA-S and may serve as a framework for clinical and epidemiological studies.
    Keywords:  Bile acid sulfates; Dispersive micro-SPE; Filtration-assisted extraction; High-throughput bioanalysis; LC–MS/MS; Surrogate matrix
    DOI:  https://doi.org/10.1016/j.talanta.2026.130028
  18. Metabolites. 2026 May 20. pii: 345. [Epub ahead of print]16(5):
      Background/Objectives: The dysregulation of thiol metabolites is strongly linked to hepatocellular carcinoma (HCC) pathogenesis. However, quantifying these highly polar and oxidation-prone thiols in clinical serum samples via conventional liquid chromatography-mass spectrometry (LC-MS) remains challenging due to their poor sensitivity and reproducibility. Methods: We developed a sensitive and robust iodoacetamine-alkyne (IAM) derivatization-based LC-MS method for quantification of seven trans-sulfuration pathway thiols in human serum. Results: IAM derivatization markedly improved the method's specificity due to enhanced chromatographic retention and diagnostic MS/MS fragments containing both the alkyne tag and analyte backbone. Sensitivity increased 33-to-160-fold versus underivatized analytes, with limits of detection of 0.02-0.1 nM. All analytes exhibited good linearity, acceptable precision with intra-day and inter-day relative standard deviations in the range of 1.2-13.8%, and high recovery from 88.6% to 102.9%. Conclusions: From the thiol quantification in human serum from 40 HCC patients and 40 healthy controls, it was found that levels of cysteine, homocysteine, glutathione, and cysteinylglycine were significantly lower in HCC patients (p < 0.05). A two-variable logistic regression model using cysteine and cysteinylglycine achieved 90.0% specificity and 80.0% sensitivity for robust HCC discrimination between HCC patients and healthy controls to some extent, with an area under the receiver operating characteristic curve of 0.88 (95% confidence interval: 0.792-0.968).
    Keywords:  chemical derivatization; hepatocellular carcinoma; iodoacetamine-alkyne; liquid chromatography-mass spectrometry; thiol metabolite
    DOI:  https://doi.org/10.3390/metabo16050345
  19. Angew Chem Int Ed Engl. 2026 May 25. e4721868
      Elucidating tissue architecture and function necessitates lipid analysis that is both spatially comprehensive and achieved at high resolution while maintaining spatial context. Conventional mass spectrometry imaging (MSI) techniques typically perform MS/MS analysis sequentially for individual lipids, creating a significant trade-off between spatial resolution and analytical depth. To circumvent this limitation, we introduce a deconvolution-based structure-specific spatial lipidomics (DeconS2L) method for coupling with per-pixel and broadband MS/MS sampling. DeconS2L capitalizes on the spatial and compositional heterogeneity inherent to all biological tissues for large-scale lipid annotation and lipidome imaging. To facilitate rapid imaging throughput and improved sample usage, lipids per pixel are co-fragmented to generate convolved MS/MS spectra. DeconS2L analysis of ∼4000 pixels in a mouse cerebellum tissue yielded 100 annotated lipids and allowed multiplexed MS/MS imaging of the tissue lipidome. Furthermore, applying DeconS2L to human hepatocellular carcinoma (HCC) tissues effectively resolved isobaric interferences that obscured tumor margins in conventional imaging. It successfully revealed the tumor-specific enrichment of odd-chain lipid PC 33:1 and distinct spatial heterogeneity in triglyceride saturation, correlating with metabolic reprogramming in HCC. DeconS2L represents a versatile methodology that effectively integrates molecular annotation with spatial lipidomics, demonstrating significant potential for in-depth biomarker discovery in clinical pathology.
    Keywords:  mass spectrometry imaging; spatial lipidomics; spectrum deconvolution; tissue heterogeneity
    DOI:  https://doi.org/10.1002/anie.4721868
  20. Bioanalysis. 2026 May 27. 1-14
      Anabolic-androgenic steroids (AAS) are widely used in clinical practice but are also frequently misused, necessitating reliable analytical methods for their detection in biological and environmental matrices. This review summarizes recent developments in analytical techniques for AAS determination between 2011 and 2024, with emphasis on chromatographic and mass spectrometric approaches. Relevant literature was identified through searches of major scientific databases, including Scopus, Web of Science, and PubMed, using keywords related to anabolic-androgenic steroids and analytical detection methods; studies were selected based on relevance to methodological advances. Gas chromatography-mass spectrometry (GC-MS) remains a robust platform, with high-resolution GC-HRMS enhancing selectivity and retrospective analysis capabilities. Liquid chromatography-mass spectrometry (LC-MS/MS) provides high sensitivity and throughput for targeted quantification, while LC-HRMS enables broader screening through suspect and non-targeted approaches. Advances in sample preparation, including microextraction and automated workflows, have improved analytical efficiency and reduced matrix effects. Emerging platforms such as ion mobility spectrometry and ambient ionization methods offer rapid screening but remain complementary to established techniques. Overall, modern AAS analysis reflects a shift toward integrated analytical strategies combining targeted and high-resolution approaches to address increasing analytical complexity and evolving regulatory demands.
    Keywords:  Anabolic–androgenic steroids; LC–MS/MS; analytical chemistry; biological matrices; chromatographic techniques; environmental analysis; high-resolution mass spectrometry; mass spectrometry; sample preparation
    DOI:  https://doi.org/10.1080/17576180.2026.2676685
  21. Toxics. 2026 Apr 24. pii: 364. [Epub ahead of print]14(5):
      Per- and polyfluoroalkyl substances (PFASs) are a large class of thousands of synthetic organofluorine chemical compounds used for many industrial applications. Humans are exposed to PFASs mainly through diet and contaminated drinking water. Studies show that PFASs induce several adverse effects on humans. A great number of human biomonitoring studies have been widely conducted with the aim of estimating exposure to PFASs. The matrices mainly investigated are blood, serum and breast milk. However, in many cases, the need for non-invasive sampling methods with a minimal impact on donors has become paramount to comply with modern ethical standards and regulations. For this reason, we developed a streamlined and efficient method for the analysis of eight perfluorocarboxylic and perfluorosulfonic acids (PFHpA; PFHxS; PFOA; PFHpS; PFNA; PFOS; PFDA; and PFUdA) in human urine samples by UPLC chromatography tandem mass spectrometry. Chromatographic and MS parameters were optimized; the method was validated for: repeatability (<20%), within-lab reproducibility (<20%), trueness (within the set 20% variation limit of agreement between the mean of the data set and the true value), efficiency (51-97%), linearity (R2 > 0.99), limits of detection (0.0003 ng/mL), and limits of quantification (0.001 ng/mL). To our knowledge, this is the first published method in Italy for the detection of PFASs in human urine.
    Keywords:  Per- and polyfluoroalkyl substances (PFAS); analytical method validation; human urine
    DOI:  https://doi.org/10.3390/toxics14050364
  22. Metabolites. 2026 May 21. pii: 348. [Epub ahead of print]16(5):
      Background: Mass spectrometry imaging (MSI) enables in situ mapping of metabolite distributions within tissues, and spatial clustering is a key step for delineating metabolically distinct regions. Nevertheless, spatial clustering methods have not been systematically benchmarked for spatial metabolomics data. Methods: Here, we evaluated the effects of ion filtering and clustering method selection on clustering performance and established a dual-metric framework that jointly assesses the spatial continuity of cluster labels and inter-cluster metabolic heterogeneity. We benchmarked 30 clustering algorithms across 12 heterogeneous MSI datasets spanning three major ion sources, four mass analyzers, and multiple spatial resolutions, covering approaches from non-spatial methods to advanced spatially aware models. Results: Noise filtering markedly improved the spatial continuity of results generated by non-spatial methods (mean improvement, approximately 28%) but provided limited benefit for spatially aware methods. Across the 12 datasets, a median of only 11 methods satisfied both evaluation criteria simultaneously, whereas SSC and DRSC met the dual-metric thresholds in at least nine datasets. In the mbrain2_pos50 dataset, the top-ranked method based on the composite dual-metric score achieved 22% higher concordance between cluster assignments and cell-type annotations than the lowest-ranked method. Conclusions: Together, the proposed evaluation framework and the online platform SMcluster provide a standardized resource for benchmarking and selecting MSI clustering methods. Our results highlight the critical roles of preprocessing and method selection in determining spatial clustering performance and offer practical guidance for spatial metabolomics studies.
    Keywords:  clustering benchmarking; inter-cluster heterogeneity; mass spectrometry imaging; online platform; spatial continuity; spatial metabolomics
    DOI:  https://doi.org/10.3390/metabo16050348
  23. Metabolomics. 2026 May 24. pii: 79. [Epub ahead of print]22(3):
      Background There is no consensus on how to interpret the large number of unknown features in untargeted metabolomics, which are sometimes referred as the "dark matter". Are these features real compounds or artifacts? Understanding this problem is critical to the annotation and interpretation of metabolomics data and future development of the field. Methods We propose a "detectable khipu" model here, to show that compounds exhibit ion group patterns that depend on their abundance. We apply this model to a systematic analysis of 61 representative public datasets from blood LC-MS metabolomics, the most common data type in biomedical studies. Results The results indicate that majority of abundant features have identifiable ion patterns, and in-source fragments contribute to less than 10% of features. Each dataset detects 1 ~ 2,000 high confidence compounds, over half of which are unknown. Conclusion The major knowledge gap in LC-MS metabolomics is therefore not the methods of grouping ions or counting fragments, but the identification of unknown compounds.
    DOI:  https://doi.org/10.1007/s11306-026-02456-y
  24. Ther Drug Monit. 2026 May 29.
       BACKGROUND: Propofol is a widely used anesthetic agent, yet it exhibits substantial interindividual variability in dosage requirements-likely driven by pharmacokinetic and/or pharmacodynamic factors. Despite ongoing efforts, accurate population-based pharmacokinetic-pharmacodynamic dosing algorithms, targeting plasma propofol concentrations, remain suboptimal. Reliable, high throughput and cost-efficient bioanalytical methods are required to quantify plasma propofol concentrations in large-scale clinical pharmacokinetic-pharmacodynamic studies to improve these algorithms. A review of the existing literature indicated an absence of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays suitable for this purpose.
    METHODS: The authors developed, optimized, validated, and clinically applied a new bioanalytical method using protein precipitation and LC-MS/MS quantification.
    RESULTS: The assay demonstrated intra- and inter-run assay accuracy of 86%-114% and 94%-105%, respectively, across the quantification range of 25-5000 ng/mL. Intra- and inter-run coefficients of variation were 2.7%-15.7% and 1.1%-5.0%, respectively. Propofol extraction recovery was 92%, with a total run-time of 5.5 minutes. This method demonstrated acceptable performance for dilution integrity, matrix effects, and stability under bench-top, freeze-thaw, autosampler, and long-term storage conditions, as well as reinjection reproducibility. This method was subsequently applied to quantify plasma propofol concentrations in a patient undergoing major noncardiac surgery, assessing the corresponding concentration-time profile. All measured propofol concentrations (648-4356 ng/mL) were within the validated assay quantification range.
    CONCLUSIONS: The authors report a robust and efficient LC-MS/MS assay for propofol that was optimized, validated, and shown to be suitable for large-scale clinical pharmacokinetic studies. This fit-for-purpose method supports population pharmacokinetic modelling to inform more individualized dosing strategies.
    Keywords:  clinical plasma assay; liquid chromatography–tandem mass spectrometry; propofol
    DOI:  https://doi.org/10.1097/FTD.0000000000001474
  25. Med Res Rev. 2026 May 25.
      Mass spectrometry imaging (MSI) has emerged as a transformative technology in pharmaceutical research, offering unprecedented capabilities to visualize drug distribution, metabolism, and target engagement in biological tissues. By combining the molecular specificity of mass spectrometry with spatial imaging resolution, this label-free approach enables simultaneous mapping of drugs, metabolites, and endogenous molecules across tissue sections, providing comprehensive insights into drug absorption, distribution, metabolism, excretion, and toxicity properties. Recent technical advances have dramatically enhanced MSI capabilities, achieving spatial resolutions down to the cellular level. The integration of tandem mass spectrometry, ion mobility separation, and dedicated data analysis tools powered by artificial intelligence has further expanded the analytical power of MSI, enabling robust molecular identification and pattern recognition in complex biological matrices. MSI applications span critical areas of drug development, from characterizing blood-brain barrier permeability and CNS drug distribution to mapping tumor microenvironment heterogeneity and evaluating anticancer drug penetration. This technology has proven invaluable for assessing drug disposition in the heart, liver, kidney, lung, as well as the gastrointestinal tract and skin. As drug research increasingly embraces multimodal approaches and single-cell analysis, MSI continues to evolve as an indispensable tool for understanding drug behavior in complex biological systems, ultimately accelerating drug development and reducing clinical failures.
    Keywords:  drug metabolism; mass spectrometry imaging; pharmacokinetics; tissue imaging
    DOI:  https://doi.org/10.1002/med.70061
  26. Toxics. 2026 May 12. pii: 422. [Epub ahead of print]14(5):
      Ultrashort-chain (USC) per- and polyfluoroalkyl substances (PFAS) are highly polar, mobile, and persistent emerging pollutants. While the environmental distribution of USC species is well-documented, their presence in widely consumed beverages remains under-characterized due to the analytical difficulty of capturing such highly polar species. This study established a robust workflow for the simultaneous determination of C1 to C14 perfluoroalkyl carboxylic and sulfonic acids, alongside other PFAS classes, in diverse beverage matrices including teas and fruit juices. Chromatographic separation was achieved using a mixed-mode inert-coated alkyl-phase LC column to enhance USC retention while maintaining performance for longer-chain analytes. A high-throughput, minimal-handling sample preparation was optimized to mitigate matrix effects and contamination. Method performance was evaluated using fortified beverage samples across 2-500 ng/L, with calibration ranges of 1-2000 ng/L and incorporation of 13 isotopically labeled internal standards. Results demonstrated acceptable accuracy (recoveries within 30% of nominal values) and optimal precision (%RSD < 12%). Application to commercial samples revealed frequent PFAS occurrence, specifically highlighting the prevalence of previously overlooked USC species in the human diet. These results demonstrate that ready-to-drink beverages are a significant pathway for human exposure, necessitating the inclusion of USC compounds in future food safety monitoring and risk assessments.
    Keywords:  beverage; juice; per- and polyfluoroalkyl substances (PFAS); tea; ultrashort-chain
    DOI:  https://doi.org/10.3390/toxics14050422
  27. Methods Protoc. 2026 May 02. pii: 70. [Epub ahead of print]9(3):
      Intra-articular soft connective tissues such as synovium and adipose tissue play a crucial role in governing joint homeostasis and disease progression in various forms of arthritis. In the knee, like many synovial joints, adipose tissue forms an integrated anatomic and functional unit with the joint-lining synovium, and the most prominent adipose depot is the infrapatellar fat pad (IFP). With growing evidence that lipid profiles in the synovium-IFP unit shift during progression of joint diseases like osteoarthritis (OA), there is strong impetus for consistent tissue collection approaches and reproducible subsequent lipid characterization. Here, we present a standardized dissection and low-input untargeted lipidomics workflow optimized for mouse knee synovium and IFP, to enable comprehensive lipid profiling. Synovium/IFP from multiple joints are pooled to increase input mass and guarantee robust lipid yield, followed by lipid extraction and high-resolution liquid chromatography-mass spectrometry (LC-MS) acquisition for global, untargeted lipidomic profiling. The analysis workflow encompasses robust feature detection, accurate lipid annotation, data transformation and normalization. These steps enhance comparability across samples, particularly those with low input amounts, while minimizing technical variance and batch effects. Using this approach, we detect a broad spectrum of lipid species spanning the major lipid categories. As expected for untargeted discovery, a subset of non-lipid species is also observed. This protocol provides a practical framework for robust, reproducible lipidomics in murine intra-articular soft tissues to support future disease-specific biomarker and drug target discovery in OA and other joint diseases.
    Keywords:  infrapatellar fat pad; lipidomics; mass spectrometry; osteoarthritis; synovium
    DOI:  https://doi.org/10.3390/mps9030070
  28. Toxins (Basel). 2026 May 08. pii: 222. [Epub ahead of print]18(5):
      Cereulide is a heat-stable cyclic depsipeptide toxin produced by Bacillus cereus and is responsible for foodborne emetic syndrome. Recent reports of Bacillus cereus contamination and cereulide occurrence in infant formula have raised increasing food safety concerns. Due to the immature immune and metabolic systems of infants, exposure to cereulide through contaminated formula may lead to potential health risks. However, direct application of existing cereulide analytical methods to infant formula remains challenging because of the unique processing technologies, encapsulated nutrients, and variable matrix composition of this product category, which may hinder toxin release and cause significant matrix interference. In practical analysis, inter-laboratory comparisons revealed that existing methods exhibited relatively large deviations and insufficient sensitivity, making them not specifically optimized for infant formula matrices. The present study was motivated by the need for a matrix-specific, sensitive, and reliable analytical method for cereulide determination in infant formula. In this study, a method based on solid-phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) was developed and validated. To improve the applicability of cereulide analysis to infant formula, this method incorporates a hydration-assisted extraction step tailored to infant formula, which increased the detected cereulide response by approximately fourfold, together with optimized SPE clean-up and improved chromatographic conditions to reduce matrix effects and enhance quantitative reproducibility. The method showed good linearity (0.1-10 μg·L-1, R2 > 0.999), low values for limit of detection (LOD) (0.03 μg·kg-1) and limit of quantification (LOQ) (0.1 μg·kg-1), and acceptable recoveries (94.4-110.3%) with RSDs below 3.7%. The developed method was successfully applied to commercial infant formula samples, and cereulide-positive samples were identified. This method provides a reliable analytical tool for the monitoring of cereulide in infant formula and contributes to improved food safety surveillance and exposure risk assessment.
    Keywords:  SPE; UPLC-MS/MS; cereulide; infant formula
    DOI:  https://doi.org/10.3390/toxins18050222
  29. Sci Rep. 2026 May 26.
      A comprehensive ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method was developed and validated for the simultaneous detection and quantification of 171 pesticide residues in wildlife blood samples. The method employed a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction procedure, optimized for efficient recovery of analytes while minimizing matrix interferences. Chromatographic separation was achieved by optimizing the mobile phase, flow rate, and gradient programme, which significantly enhanced peak separation and stability. The mass spectrometric analysis was performed using a full scan-ddMS² mode in an Orbitrap mass spectrometer, with three intense precursor-to-fragment ion transitions for each pesticide. The method was validated according to SANTE guidelines, assessing linearity, recovery, precision, accuracy, matrix effect and limit of quantification (LOQ). The calibration curves exhibited strong linearity (R² = 0.990-0.999) over the concentration range of 1-250 µg L⁻¹. Mean recoveries for 109 pesticides ranged from 70% to 119% at 25 µg L⁻¹ spiking level, with relative standard deviations (RSD) below 20% for most compounds. LOQs ranged from 5.0 to 59.64 µg L⁻¹. The matrix effect was evaluated and addressed through matrix-matched calibration. The developed UHPLC-HRMS method enables sensitive, selective, and reliable quantification of a wide range of pesticides in blood, supporting long-term monitoring of pesticide exposure in wildlife and aiding conservation efforts.
    Keywords:  Blood matrix; Method validation; Pesticide residues; QuEChERS extraction; UHPLC-HRMS
    DOI:  https://doi.org/10.1038/s41598-026-49063-4
  30. Anal Chem. 2026 May 28.
      Modified DNAs and RNAs often exist in trace amounts, which makes detection challenging. Adding ionizable groups to nucleobases (NBs) can enhance sensitivity in liquid chromatography-tandem mass spectrometry (LC-MS2). Traditional derivatization methods for modified nucleosides require complex preparation under harsh conditions and cover only limited modifications. We present an ultrafast (microseconds) derivatization technique using ionizable imidazole, facilitated by microdroplets through supersonic electrospray-induced aldehyde condensation for direct LC-MS2 analysis. This technique universally tags the exocyclic amine (-NH2) or pyrrolic nitrogen (-NH) of five NBs without base damage, particularly guanosine, which is prone to oxidation. Sensitive modifications located on ring carbon, like oxidative lesions and epigenetic marks, or nontagging sites of ring nitrogen, like N-alkylation on guanosine, and depurinating adducts, remain intact. The rapid derivatization avoids artifact generation and allows online postcolumn in situ detection, maintaining chromatographic separation. This method achieves over 90% derivatization ratio and improves quantification sensitivity by more than 10-fold. It also enables a quick test for tagging site-blocked modifications. We demonstrate this method by locating stable adduction sites of 4-hydroxy-estradiol (4OHE2) in chromatin DNA and quantifying 4OHE2-induced oxidation and released adducts in MCF-7 cells, achieving 13-43-fold higher sensitivity with significantly reduced sample amounts. Key parameters influencing detection efficiency were systematically explored for routine application.
    DOI:  https://doi.org/10.1021/acs.analchem.6c00004
  31. Anal Sci. 2026 May 25.
      The field desorption (FD) method is a soft ionization technique that can analyze nonvolatile, thermally labile compounds with minimizing fragmentation. While advantageous for analyzing viscous materials like detergents, FD traditionally lacks chromatographic separation, making it challenging to distinguish stereoisomers such as D-sorbitol and D-mannitol, which have identical molecular masses. Additionally, FD has historically been limited to qualitative analysis due to manual sampling variability and volatilization under high vacuum. This study demonstrates a novel approach to overcome these limitations. Researchers synthesized deuterated D-sorbitol and D-mannitol to serve as internal standards for quantitative analysis. Using a ruthenium on activated carbon (Ru/C) catalyst in D2O, they achieved 98% deuterium content at 80 °C. These internal standards proved essential for achieving acceptable recovery rates (108-117%) in model detergents, particularly at higher concentrations. A significant breakthrough in this research is the first-ever demonstration of stereoisomer separation using the FD method alone. By reducing the emitter's rising current rate from 51.2 mA/min to 3.2 mA/min, the researchers were able to separate sorbitol and mannitol based on their different retention times. This separation is attributed to differences in the compounds' physicochemical properties, such as crystallinity and solubility, which influence their desorption energy from the emitter surface. Compared to electrospray ionization (ESI), which suffered from matrix effects and instability for sugar alcohol analysis, the FD method provided a more direct and reliable quantitative route. Although current FD instruments are not yet optimized for precise current control, this study establishes FD as a valuable tool for quality control in detergent production, offering both isomer separation and direct quantification without complex derivatization.
    Keywords:  Deuteration; FD-MS; Internal standard; Stereoisomer separation; Sugar alcohols
    DOI:  https://doi.org/10.1007/s44211-026-00927-9
  32. Metabolomics. 2026 May 29. pii: 90. [Epub ahead of print]22(3):
       INTRODUCTION: Human breast milk is the optimal source of nutrition for newborns and contains hundreds of bioactive compounds that influence infant health and development. Characterizing its composition at scale is important for understanding how maternal diet, health status, and other factors affect milk quality. However, manual NMR profiling methods are too slow for large cohort studies. MagMet is a program designed for the rapid, automated processing and profiling of 1D 1H NMR spectra from complex mixtures of small molecules. In this study, we develop a version of MagMet (called MagMet-HM) capable of rapid, quantitative automated NMR analysis of human breast milk.
    METHODS: A library consisting of 72 metabolites was created based on the literature and comparison with experimental NMR spectra, that are known to be abundant or consistently detectable in breast milk. NMR spectra of ultrafiltered breast milk was then used to optimize and validate the performance of MagMet-HM in the automated NMR analysis of human breast milk.
    RESULTS: Performance was benchmarked against manual profiling using Chenomx (version 8.3), with median and mean absolute percent errors of approximately 5.1% and 9.1%, respectively. MagMet-HM completes profiling in 10 min (on a single CPU) which is 3-6 times faster than manual methods.
    CONCLUSIONS: MagMet-HM offers a convenient, fast, and accurate method for the high-throughput metabolomic profiling of human breast milk. MagMet-HM is available at https://www.magmet.ca .
    Keywords:  Automated spectral profiling; Human breast milk; Human milk oligosaccharides; Metabolomics; Milk metabolome; NMR spectroscopy
    DOI:  https://doi.org/10.1007/s11306-026-02472-y
  33. Rapid Commun Mass Spectrom. 2026 Aug 30. 40(16): e70115
       RATIONALE: Organophosphate esters (OPEs) have emerged as dominant flame retardants following the phase-out of brominated alternatives, highlighting the need for accurate quantification due to their associated toxicity. Comprehensive analysis of OPEs, however, is challenging due to the wide polarity range of congeners and issues with co-elution. To address these challenges, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of 17 OPEs.
    METHODS: Using an ACQUITY UPLC BEH C18 column with sub-2-μm particles, we achieved baseline separation of critical isomer pairs, including TPrP/TiPP and TBP/TiBP. Furthermore, a novel dual-solvent sequential elution strategy, employing methanol and ethyl acetate for solid-phase extraction (SPE), significantly improved recovery rates.
    RESULTS: Recovery of the highly polar TMP increased from 14.0% to 33.2%, while that of the hydrophobic TEHP improved from 41.0% to 103.2%. This dual-solvent approach enabled quantitative recovery (33.2%-153%) for all 17 OPEs, compared to successful recovery of only six compounds using traditional single-solvent protocols. The method demonstrated detection limits ranging from 0.01 to 2.61 ng/L, with 82% of compounds achieving limits below 0.2 ng/L. Validation across three concentration levels (4-40 ng/L) yielded mean recoveries of 73.5%-109%, with relative standard deviations (RSDs) ranging from 1.0% to 13.0%. Application of this method to surface water and wastewater treatment plant (WWTP) samples detected 11 OPEs at concentrations ranging from 0.27 to 11.41 ng/L, with TBEP exhibiting a 100% detection frequency. WWTP removal efficiencies demonstrated property-dependent trends: hydrophobic compounds (log Kow > 3.5) were removed by > 99%, whereas hydrophilic OPEs, such as TMP and TCEP, showed limited removal, with 88%-100% persistence.
    CONCLUSION: This newly developed method provides a robust analytical foundation for environmental monitoring and risk assessment of OPEs.
    Keywords:  UPLC–MS/MS; isomer separation; organophosphate esters; solid‐phase extraction
    DOI:  https://doi.org/10.1002/rcm.70115
  34. J Sep Sci. 2026 Jun;49(6): e70455
      Golvatinib is a tyrosine kinase inhibitor in development for treating solid tumors such as advanced hepatocellular carcinoma. The present study aimed to clarify the metabolic fates of golvatinib using human and rat liver microsomes. For this purpose, a comprehensive and systematic workflow was established for investigating the in vitro metabolic fates. Separation was performed on a Waters ACQUITY BEH C18 column with a mobile phase consisting of 0.1% formic acid in water and acetonitrile. Detection was carried out by multiple reaction monitoring mode using the transitions m/z 634.3→184.2 for golvatinib and m/z 650.3→200.2 for golvatinib N-oxide. This method exhibited excellent linearity across a concentration range of 1.0-2000 nM (r > 0.995). The validated assay was subsequently applied to evaluate the metabolic stability of golvatinib. The results revealed that golvatinib underwent rapid metabolism in rat liver microsomes (t1/2 = 13.76 ± 0.79 min), while its turnover was considerably slower in human liver microsomes (t1/2 = 56.93 ± 3.17 min). Furthermore, twelve metabolites were identified using Orbitrap high-resolution mass spectrometry (HRMS) via accurate mass determination and fragmentation analysis. Major metabolic pathways of golvatinib encompass oxygenation, N-demethylation, N-oxidation, N-hydroxylation, dealkylation, oxidative deamination, and oxidative defluorination. Further study demonstrated that CYP3A4 was the principal enzyme involved in metabolizing golvatinib. To the best of our knowledge, this is the first report combining ultra-high-performance liquid chromatography-tandem MS (UHPLC-MS/MS) with UHPLC-Quadrupole-Orbitrap-HRMS for profiling golvatinib metabolism in vitro, thereby laying a foundation for subsequent pharmacokinetic study.
    Keywords:  cytochrome p450 3a4; golvatinib; liver microsomes; metabolic stability; metabolite identification
    DOI:  https://doi.org/10.1002/jssc.70455
  35. Mikrochim Acta. 2026 May 29. pii: 424. [Epub ahead of print]193(6):
      A novel integrated ion source component based on molecularly imprinted polymer-coated metal-organic cages (SSS@MOC@MIP) was developed for rapid aflatoxin (AFs) detection via ambient mass spectrometry (AMS). By leveraging the synergistic effects of a high-surface-area MOC scaffold which endows the material with exceptional adsorption capacity and specific imprinted cavities, this integrated probe enables simultaneous enrichment, purification, and ionization on a single device. The method effectively suppressed severe matrix interferences from peanut oil and corn starch (matrix effects within ± 15.3%) and achieved a high imprinting factor of 3.27. Under optimized conditions, the SSS@MOC@MIP-AMS method allowed for rapid quantification within 1 min, with low limits of detection (0.11-0.54 µg/kg) and satisfactory recoveries (79.05%-108.29%), and excellent precision with relative standard deviations (RSDs) less than 14.63%. The probe exhibited robust reusability for at least six cycles. Practical application to commercial samples confirmed its reliability, offering a powerful and high-throughput tool for the rapid screening of trace-level aflatoxins in complex food matrices.
    Keywords:  Aflatoxins; Ambient mass spectrometry; Food safety; Metal-organic cage; Molecularly imprinted polymer
    DOI:  https://doi.org/10.1007/s00604-026-08128-x
  36. Anal Bioanal Chem. 2026 May 27.
      Atypical myopathy (AM) is a frequently fatal, toxin-induced disease in horses, characterised by acute progression and mortality rates exceeding 70% within 72 h. The disease is caused by ingestion of seedlings or seeds of certain maple species that contain the toxins hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG). Early diagnosis depends on reliable detection of these compounds and their metabolites. However, most available LC-MS/MS assays are limited by derivatisation requirements and dependence on serum and urine matrices. Here, a rapid derivatisation-free flow injection analysis (FIA)-tandem mass spectrometry (MS/MS) method was developed for the simultaneous quantification of nine analytes: HGA, methylenecyclopropylacetyl-carnitine (MCPA-carnitine), and seven diagnostically relevant acylcarnitines, from dried blood spots (DBS). The procedure requires only 50 µL of whole blood, minimal sample preparation, and enables results within one minute. The assay provides low limits of quantification (0.01-0.1 μmol/L) with excellent linearity (R2 > 0.994), exhibiting analytical performance suitable for rapid screening of AM biomarkers. Moreover, analytes showed adequate stability in DBS during short-term storage at ambient temperature and prolonged storage at -20 °C. Clinical performance of the workflow was evaluated using 17 DBS samples from horses with AM and 15 control DBS samples. AM cases were defined based on the high specificity of HGA and MCPA-carnitine, with accompanying acylcarnitine profiles reflecting disease-associated metabolic disruption. Over 2 years, more than 250 DBS samples from horses suspected of AM were routinely analysed. Overall, the results establish a robust, high-throughput FIA-MS/MS platform integrating DBS sampling with non-derivatised analysis, offering a practical screening tool for remote sample collection, efficient transport, and multi-analyte diagnosis of AM.
    Keywords:  Dried blood spot sampling; Equine atypical myopathy ; Flow injection analysis-tandem mass spectrometry; Hypoglycin A; MCPA-carnitine; Screening
    DOI:  https://doi.org/10.1007/s00216-026-06566-3
  37. Antibiotics (Basel). 2026 Apr 30. pii: 459. [Epub ahead of print]15(5):
      Background: Phospholipids are essential components of bacterial membranes and play central roles in membrane integrity and adaptation to antibiotic stress. However, confident annotation of phospholipid molecular species remains challenging due to the complexity of the lipidome and the limited structural constraints in conventional lipidomics workflows. Methods: Here, we present a bacterial phospholipidomic framework that integrates orthogonal structural evidence to achieve high-confidence and traceable annotation. Thin-layer chromatography (TLC) provides phospholipid headgroup assignment, gas chromatography-mass spectrometry (GC-MS) defines the acyl-chain pool, and Paternò-Büchi derivatization enables C=C localization, collectively restricting the structural search space prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A rule-based ion prediction library further standardizes diagnostic ion assignment and reduces annotation ambiguity. Results: Applying this platform, we found Escherichia coli in the stationary phase remodeled the membrane phospholipids, with cardiolipin (CL) increasing from ~5% to ~10% and cyclopropane-containing phospholipid species rising to ~75%. Similar remodeling patterns are observed under diverse antibiotic exposures at sub-inhibitory concentrations, consistent with convergence toward a tolerance-associated membrane state. Extension of the framework to Enterococcus faecium supports proof-of-concept application in an additional Gram-positive model, with vancomycin-resistant strains exhibiting pronounced phosphatidylglycerol (PG) enrichment and reduced CL. Conclusions: Our work provides a scalable and reproducible strategy for bacterial phospholipid annotation, enabling molecular-species-resolved investigation of membrane adaptation and offering a framework for future exploration of lipid homeostasis pathways as potential antimicrobial targets.
    Keywords:  MS/MS; Paternò–Büchi derivatization; antibiotic tolerance; bacterial phospholipid profiling; membrane remodeling
    DOI:  https://doi.org/10.3390/antibiotics15050459
  38. Electrophoresis. 2026 May 23.
      Mycosporine-like amino acids (MAAs) are photoprotective compounds primarily produced by marine organisms, especially red algae. Chemically, MAAs and their precursors are low-molecular-weight natural products that absorb ultraviolet radiation in the range of 270-360 nm without generating free radicals. Owing to these properties, MAAs have attracted considerable interest for potential pharmaceutical and cosmetic applications as natural sunscreen agents. In this study, the first hydrophilic interaction liquid chromatography-ultra-performance liquid chromatography (HILIC-UPLC) method, coupled with both diode array detector (DAD) and tandem mass spectrometry (MS/MS), was developed for the determination of 11 MAAs and 2 MAA precursors in various algal species. As column, a YMC-Triart Diol-HILIC (particle size: 1.9 µm) was used, and the separation of the MAAs realized in under 5 min. The validation of the method was carried out following International Council for Harmonisation (ICH) guidelines, demonstrating linearity, selectivity, precision, and accuracy. The analysis of extracts prepared from well-known species, such as Porphyra sp. and Chondrus crispus, confirmed practical applicability. Demonstrating feasibility of MS/MS-based quantification of co-eluting compounds, the method represents an appealing alternative that surpasses established procedures not only in terms of analysis time but also by providing a substantially increased informative value.
    Keywords:  hydrophilic interaction liquid chromatography (HILIC); marine organisms; mycosporine‐like amino acids (MAAs); ultra‐performance liquid chromatography (UPLC)
    DOI:  https://doi.org/10.1002/elps.70110
  39. J Am Soc Mass Spectrom. 2026 May 28.
      Mass spectrometry imaging (MSI) enables spatially resolved molecular analysis, but automatic data reduction imposed by instrument software, such as in Orbitrap systems, can severely limit the detection of low-abundance ions. When these ions are targeted, the resulting sparse detection hinders spatial interpretation and compromises quantitative accuracy. Using an external high-performance data acquisition system to capture full-length transients, we demonstrate that adjacent-pixel averaging of full-profile (unreduced) Orbitrap data markedly improves the detection frequency of low-abundance peaks without affecting the detection of highly abundant species in biological tissue samples. Pixel averaging increased the coverage of low-abundance ions from less than 10% to over 90% in mouse brain and nonhuman primate vaginal tract tissues. Quantitative accuracy was also enhanced, as shown by improved agreement between observed and theoretical isotopic ratios, and between MSI-derived and liquid chromatography (LC)-MS/MS measured total analyte concentrations in adjacent tissue sections. These findings establish pixel averaging as a simple yet powerful strategy to extend MSI sensitivity and improve quantitative biochemical mapping in complex tissues, while remaining fully compatible with other sensitivity-enhancing techniques.
    Keywords:  MALDESI; mass spectrometry imaging; sensitivity enhancement
    DOI:  https://doi.org/10.1021/jasms.6c00081