bims-metlip 2026-08-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–08–23
thirty-six papers selected by
Sofia Costa, Matterworks

Quantitative Integration of Targeted and Nontargeted Metabolomics Using Multi-Stable Isotope Chemical Tagging with UHPLC-QToF MS: Application to Myeloid Leukemia Metabolism.
A protocol for untargeted metabolomics using anion-exchange chromatography and high-resolution mass spectrometry.
Development and validation of dispersive liquid-liquid microextraction coupled with LC-MS/MS for plasma 5-hydroxyindoleacetic acid quantification with application as a biomarker in major depressive disorder.
LC-MS-Based Analytical Strategies for Ganglioside Profiling.
A validated bioanalytical LC-MS/MS method for the simultaneous determination of molnupiravir, N-hydroxycytidine, and baricitinib in human plasma.
Validation and Clinical Evaluation of an LC-MS/MS Method for Serum Full-Length Parathyroid Hormone 1-84 with Application to Chronic Kidney Disease.
Protocol for untargeted multi-omics dried blood spot extraction using liquid chromatography-tandem mass spectrometry.
Target quantification and ion mobility-HRMS suspect screening of BPA and its alternatives in human urine.
Analysis of Ganglioside Molecular Species Focusing on Ceramide Structures Using Thin-Layer Chromatography-Mass Spectrometry.
An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of gentamicin in human serum.
Optimizing Oxylipin Analysis with Liquid Chromatography Mass Spectrometry through Bio-Inert Systems and Ion Funnel Adjustments.
Development and validation of an HPLC-MS/MS method for the quantification of Aficamten in human plasma and urine and application to a clinical pharmacokinetic study.
Spatially Resolved Metabolomics of Optimal Cutting Temperature (OCT) Compound-Embedded Tumors.
Decoding the urinary metabolome of aromatic amino acid pathways in Alport syndrome.
Simultaneous quantification of seventeen kratom alkaloids and metabolites in human plasma and its application to clinical pharmacokinetic sample analysis.
Resolving Spectral Complexity in 4D Lipidomics Using a Two-Dimensional Deconvolution Framework.
Mass Spectrometry-Based Untargeted Metabolite Profiling to Discover Molecular Consequences of Mammalian Cell Infection with Fusobacterium nucleatum.
New chromatographic and data evaluation approaches for the comprehensive investigation of metal-phytosiderophore-complex dynamics.
Validation of an integrated analytical method for the simultaneous quantification of 12 PFAS in drinking water, plant-based milk alternatives and skimmed milk.
Determination of 13 per- and polyfluoroalkyl substances in human plasma samples using LC-MS/MS: application to capillary microsamples.
Development and validation of a multiplexed LC-HRMS method for nine GLP-1 receptor agonists and its pharmaceutical application.
Targeted metabolomics of postmortem human cardiac tissue using the Biocrates MxP Quant 500 kit.
Emerging analytical techniques for tamoxifen and metabolite quantification in diverse matrices.
Multi-omic analyses of the same sample using metabolomics, lipidomics, proteomics, phosphoproteomics, and glycoproteomics.
Structure Analysis of Gangliosides: Mass Spectrometry Imaging.
Analytical strategies for complex injectables: the expanding role of LC-MS technologies.
A systematically evaluated SEC-ICP-MS/MS workflow for Mn, Fe, Cu, and Zn speciation in human serum.
Development and validation of a cost-effective and reliable HPLC-FLD method for cortisol determination in human hair.
Mass spectrometry similarity analysis and pattern recognition-driven metabolomics strategy for species and geographic chemotyping of Gardenia fruit.
Mass Spectrometry-Based Anti-Doping Analysis: A Critical Review of Emerging Analytical and Sample Pretreatment Strategies.
Automated Tissue Classification and Candidate Biomarker Feature Extraction in Mass Spectrometry Imaging Based on Interpretable Deep Learning Algorithm.
How well can off-the-shelf LLMs elucidate molecular structures from mass spectra using chain-of-thought reasoning?
Integrating mass spectrometry imaging and data-driven segmentation for spatial metabolic mapping of diabetic eye disease.
Metal-Anchored Silicon-Based Nanostructures Enable Dual-Polarity SALDI-MS Analysis of Small Molecules.
Development and Validation of an HPLC-MS/MS Method for the Determination of Benzodiazepines in Hair. Comparison of Two Different Extractions and Applications on Real Samples.
Quantitation of Cirtuvivint (SM08502) in Human Plasma by LC-MS/MS.

Anal Chem. 2026 Aug 18. 98(32): 23906-23919

Quantitative Integration of Targeted and Nontargeted Metabolomics Using Multi-Stable Isotope Chemical Tagging with UHPLC-QToF MS: Application to Myeloid Leukemia Metabolism.

Takahiro Takayama, Taiyo Tsutsumi, Tomoya Higuchi, Satoshi Takahashi, Yoshihiro Hayashi, Koichi Inoue.

Mass spectrometry-based metabolomics is widely used for comprehensive metabolic profiling. However, most current workflows rely on relative signal intensities, which limit comparability across experiments and prevent quantitative interpretation. This limitation arises from the difficulty in estimating analyte-specific response behavior in the absence of isotopically labeled standards. In this study, we present multistable isotope chemical tagging (MUSIC) as an isotope-resolved internal calibration framework that enables the approximation of response characteristics within a single experimental design. This approach uses multiple isotope-coded tagging reagents as internal calibration points instead of conventional internal standards, enabling the construction of internal calibration curves that account for both tagging efficiency and matrix effects. Internal calibration curves were established for amine-containing metabolites using a dilution series of tagged standard mixtures, enabling robust slope estimation. The resulting calibration framework allows accurate quantification of targeted metabolites and slope-based correction of nontargeted features through reference matching. In validation experiments involving 146 metabolites in serum, the method achieved accuracy and precision within ±15% using only two analytical runs. We further demonstrate that the framework enables the consistent recovery of fold changes across samples and supports comparative metabolic analysis without relying on compound-specific labeled standards. These results establish MUSIC not only as a chemical tagging strategy but also as a quantitative measurement framework that approximates analyte-specific response characteristics for integrated targeted and nontargeted metabolomics for amine-containing metabolites. Following validation, we applied this approach to blood samples to identify the biomarkers of myeloid leukemia.

DOI: https://doi.org/10.1021/acs.analchem.6c04094
STAR Protoc. 2026 Aug 19. pii: S2666-1667(26)00437-5. [Epub ahead of print]7(3): 104784

A protocol for untargeted metabolomics using anion-exchange chromatography and high-resolution mass spectrometry.

Marius Jouan, Sylvain Henry, Andrei Turtoi.

  Polar metabolites are challenging to analyze using common reverse-phase chromatography. However, these molecules are most essential for deciphering biological phenotypes. Here, we present a protocol for polar metabolite analysis based on anion-exchange chromatography coupled with high-resolution mass spectrometry (AEC-HRMS). We outline preparation techniques for common biological matrices, instrumental setup for untargeted metabolomics, including sample analysis and essential data treatment.

Keywords:  Mass Spectrometry; Metabolomics; Protocols in Metabolomics and Lipidomics

DOI:  https://doi.org/10.1016/j.xpro.2026.104784
Sci Rep. 2026 08 16. pii: 25530. [Epub ahead of print]16(1):

Development and validation of dispersive liquid-liquid microextraction coupled with LC-MS/MS for plasma 5-hydroxyindoleacetic acid quantification with application as a biomarker in major depressive disorder.

Saleh I Alaqel, Ayman A Abd Al Maksoud, Ahmed M Aljameeli, Mashael M Alaradi, Mohammed F Aldawsari, Ahmed Serag, Atiah H Almalki.

  A sensitive dispersive liquid-liquid microextraction (DLLME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for plasma 5-hydroxyindoleacetic acid (5-HIAA) quantification as a depression biomarker. Box-Behnken design evaluated four DLLME factors (disperser volume, extraction volume, pH, centrifugation time) where second-order polynomial model identified optimal conditions with R² = 0.9444, achieving 95.1% extraction recovery. Chromatographic separation employed Poroshell 120 EC-C18 column (2.7 μm, 50 × 3.0 mm) with isocratic elution (20% acetonitrile with 0.1% formic acid) and 5-minute run time. Positive electrospray ionization with multiple reaction monitoring transitions (m/z 192.1 → 146.1 for 5-HIAA; 197.1 → 151.1 for 5-HIAA-d5) enabled selective detection. Validation following ICH M10 guidelines with surrogate matrix calibration demonstrated excellent linearity (r² = 0.9997, 5-1000 ng/mL), adequate sensitivity (LLOQ 5 ng/mL), satisfactory accuracy (-10.3% to + 0.2%) and precision (6.7-14.6% CV), minimal matrix effects (96.8-102.5%), and successful parallelism assessment confirming surrogate-authentic matrix equivalence. Clinical application revealed significantly elevated plasma 5-HIAA in depression (25.73 vs. 17.35 ng/mL, 48% elevation, p < 0.001, Cohen's d = 3.059), strong correlation with Hamilton Depression Rating Scale scores (r = 0.791, r² = 0.625), and excellent diagnostic performance (AUC = 0.985, sensitivity 88.2%, specificity 100%). This validated method demonstrates clinical utility for objective depression assessment combining microextraction sample preparation, sensitive detection, and comprehensive biomarker evaluation.

Keywords:  5-Hydroxyindoleacetic acid; Dispersive liquid-liquid microextraction; LC-MS/MS; Major depressive disorder; Surrogate matrix validation

DOI:  https://doi.org/10.1038/s41598-026-65693-0
Methods Mol Biol. 2026 ;3035 77-89

LC-MS-Based Analytical Strategies for Ganglioside Profiling.

Kazutaka Ikeda.

  Gangliosides, a class of glycosphingolipids containing sialic acid residues, are abundantly expressed in neuronal membranes and play essential roles in cellular signaling and intercellular communication. Impaired ganglioside metabolism contributes to the pathogenesis of lysosomal storage disorders and neurodegenerative diseases. The structural diversity and functional relevance of gangliosides necessitate analytical methods with high sensitivity, resolution, and specificity. In recent years, liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful tool for comprehensive ganglioside profiling. This review presents current LC-MS-based strategies for ganglioside analysis, with a particular focus on sample preparation methods, chromatographic separation techniques, and mass spectrometric approaches aimed at enabling accurate structural elucidation and pathway-level insights into ganglioside metabolism.

Keywords:  Gangliosides; Glycosphingolipids; LC–MS; Lipidomics; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-5288-6_7
Bioanalysis. 2026 Aug 16. 1-15

A validated bioanalytical LC-MS/MS method for the simultaneous determination of molnupiravir, N-hydroxycytidine, and baricitinib in human plasma.

Fatma H Ghazy, Ramzia I El-Bagary, Samar H Fahim, Sally T Mahmoud.

  A sensitive and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of the anti-COVID-19 agents molnupiravir (MPV), its active metabolite N-hydroxycytidine (NHC), and baricitinib (BARI) in human plasma. As MPV is rapidly converted to NHC in vivo, whereas BARI is predominantly eliminated unchanged, NHC was generated in vitro by alkaline hydrolysis to enable accurate determination of the active metabolite. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column using methanol and 0.1% formic acid in water (90:10, v/v). Escitalopram was selected as the internal standard owing to its consistent extraction recovery, and satisfactory chromatographic performance. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate, yielding recoveries of 87.5-91.6% for MPV, 87.5-89.4% for NHC, and 87.9-94.9% for BARI. The method was validated according to FDA bioanalytical guidelines over concentration ranges of 20-500, 20-10.000, and 20-500 ng/mL for MPV, NHC, and BARI, respectively. Accuracy, precision, stability, recovery, carryover, and matrix effect met the acceptance criteria. Normalized matrix factors for MPV, NHC, and BARI ranged from 0.948-0.984, 0.900-0.902, and 0.912-1.014, respectively, with CV values below 1%, indicating negligible matrix interference. The proposed method provides a reliable analytical tool for future pharmacokinetic and therapeutic drug monitoring studies.

Keywords:  LC-MS/MS; N-hydroxycytidine; baricitinib; human plasma; method validation; molnupiravir

DOI:  https://doi.org/10.1080/17576180.2026.2715920
ACS Pharmacol Transl Sci. 2026 Aug 14. 9(8): 2151-2161

Validation and Clinical Evaluation of an LC-MS/MS Method for Serum Full-Length Parathyroid Hormone 1-84 with Application to Chronic Kidney Disease.

Chunyan Li, Shangqi Yin, Huiru Zhao, Ruichen Liu, Xianglong Zhao, Zhao Zhang, Fan Liu, Jie Yang, Jun Wu.

  Objective: Accurate measurement of full-length parathyroid hormone (PTH) 1-84 is important for evaluating disorders of calcium-phosphate metabolism, particularly in chronic kidney disease (CKD). Because immunoassay results may vary according to assay design and analyte recognition, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for selective quantification of serum PTH 1-84 and explored its potential clinical relevance in kidney-related disease. Methods: Serum samples were processed by solid-phase extraction and spiked with an isotope-labeled internal standard. PTH 1-84 was separated on a C4 column and detected in positive electrospray ionization mode using multiple reaction monitoring. Analytical performance, including selectivity, linearity, accuracy, precision, recovery, and matrix effect, was evaluated using predefined acceptance criteria. A retrospective clinical analysis was then performed to compare this method with chemiluminescent immunoassay (CLIA) and to assess associations with calcium-phosphate metabolism markers. Results: The assay showed satisfactory analytical performance with negligible matrix effect and no meaningful interference under the tested conditions. In uremic patients, PTH concentrations measured by LC-MS/MS were generally lower than those obtained by CLIA, although the two methods remained correlated. PTH 1-84 measured by LC-MS/MS showed a stronger association with serum phosphate than with serum calcium in this cohort. In patients with renal involvement, both methods showed higher PTH concentrations than in patients without renal involvement. Conclusion: This LC-MS/MS assay provides selective quantification of serum full-length PTH 1-84 with satisfactory analytical performance. It may serve as an orthogonal analytical approach for studies of mineral metabolism in CKD-related disease, although intermethod differences should be interpreted cautiously.

Keywords:  LCMS/MS; bone disorder; chronic kidney disease; conventional immunoassays; parathyroid hormone

DOI:  https://doi.org/10.1021/acsptsci.5c00748
STAR Protoc. 2026 Aug 19. pii: S2666-1667(26)00436-3. [Epub ahead of print]7(3): 104783

Protocol for untargeted multi-omics dried blood spot extraction using liquid chromatography-tandem mass spectrometry.

Esabella R Powers, Timothy J Garrett.

  Dried blood spots (DBS), most notably employed in newborn screening, present a valuable opportunity to analyze whole blood via a stable and simplified sample collection. DBS are generally used to evaluate one class of compounds at a time. Herein, we present a protocol to semi-quantitatively evaluate metabolites and lipids from the same DBS using a multi-omics approach. We describe steps for extracting both metabolites and lipids from the paper-based sample and analyzing them via liquid chromatography-tandem mass spectrometry. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Keywords:  Health sciences; Metabolomics; Protocols in metabolomics and lipidomics

DOI:  https://doi.org/10.1016/j.xpro.2026.104783
Talanta. 2026 Aug 20. pii: S0039-9140(26)01144-6. [Epub ahead of print]312(Pt B): 130488

Target quantification and ion mobility-HRMS suspect screening of BPA and its alternatives in human urine.

Riccardo De Santo, Lucia Coppola, Fabiola Ferri, Cinzia La Rocca, Anna Laura Iamiceli.

  Bisphenol A (BPA) and its structural alternatives are contaminants of concern due to their endocrine-disrupting properties and widespread human exposure. The progressive replacement of BPA by alternative bisphenols has increased the need for sensitive analytical methods capable of monitoring both regulated and emerging analogues in biological matrices. In this study, a fast analytical approach based on ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and ion mobility-high-resolution mass spectrometry (IM-HRMS) was developed for the determination and suspect screening identification of bisphenols in human urine. Following enzymatic deconjugation and solid-phase extraction, BPA and eight priority bisphenols were quantified by UHPLC-MS/MS. Method validation demonstrated excellent linearity (r2 = 0.9976-0.9999), limits of quantification of 0.1 ng/mL, relative recoveries between 89.1 and 135.0%, and intermediate precision generally below 10.0%. Application of the method to urine samples from 25 healthy volunteers revealed widespread exposure to BPA, bisphenol S (BPS), and bisphenol F (BPF). To extend chemical coverage, the same extracts were analyzed by IM-HRMS-based suspect screening, leading to the identification of additional bisphenols, including bisphenol G (BPG) and bisphenol M (BPM). The integration of IM provided collision cross section (CCS) values and enabled the discrimination of structurally related and isobaric compounds, improving annotation confidence. Furthermore, new experimental CCS reference values were established for bisphenol E (BPE), BPG, and BPM, expanding the currently available database for bisphenol characterization. The proposed approach provides a comprehensive strategy for monitoring human exposure to both of known and emerging bisphenols and supports future biomonitoring and risk assessment studies.

Keywords:  Bisphenol a alternatives; Collision cross section; Human biomonitoring; Ion mobility mass spectrometry; Suspect screening; UHPLC–MS/MS

DOI:  https://doi.org/10.1016/j.talanta.2026.130488
Methods Mol Biol. 2026 ;3035 113-122

Analysis of Ganglioside Molecular Species Focusing on Ceramide Structures Using Thin-Layer Chromatography-Mass Spectrometry.

Noboru Hashimoto, Shizuka Ito, Akihito Yamamoto.

  Thin-layer chromatography is widely used for the analysis of glycolipids and phospholipids. Typically, after separation with various solvents, molecular species are identified by chemical detection or immunostaining. This chapter describes a method for structural analysis of molecules by directly performing mass spectrometry after chromatographic separation. We introduce a classical ganglioside analysis method and a technique for structure analysis of separated lipids using a robotic ion source for direct MS analysis.

Keywords:  Ceramide; Ganglioside; Hydroxylation; Mass spectrometry; Robotic ion source; Thin-layer chromatography

DOI:  https://doi.org/10.1007/978-1-0716-5288-6_10
Clin Chem Lab Med. 2026 Aug 19.

An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of gentamicin in human serum.

Jo Anne Wright, Neeraj Singh, Annika Brauch, Friederike Bauland, Andrea Geistanger, Judith Taibon, Elie Fux.

   OBJECTIVES: Therapeutic drug monitoring (TDM) of gentamicin is critical to ensure efficacy and minimize toxicity. We report a candidate reference measurement procedure (RMP) to quantify gentamicin in biological matrices, establishing an unbroken traceability chain to the International System of Units (SI).
METHODS: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method was validated using quantitative nuclear magnetic resonance (qNMR) spectroscopy to replace historical biological activity-based standardization with gravimetric mass. Strict ID-MS principles were applied to the major congeners (C1, C1a, and C2). Isomers C2a and C2b, eluting at distinct retention times, were quantified using C2-D3 as a structural analog internal standard. Pharmacokinetic-based analytical performance specifications set the maximum allowable standard measurement uncertainty (k=1) at ≤6.7 %.
RESULTS: The RMP demonstrated high analytical control, with trueness (bias 1.0-2.8 %) and within-run precision (CV 0.2-0.9 %) fulfilling clinical requirements. Expanded measurement uncertainties (k=2) for target value assignment ranged from 2.0 to 2.4 %. Comparison with the current JCTLM-listed RMP revealed a systematic negative bias of -9.2 %. A three-way method correlation confirmed these differences as a direct result of the metrological transition from activity to SI-traceable mass units.
CONCLUSIONS: This candidate RMP provides a robust, highly precise SI-traceable foundation for gentamicin quantification. While the resulting downward shift in reported concentrations may necessitate re-evaluating established clinical decision thresholds and TDM protocols, the clinical requirement for such adjustments remains to be fully determined.

Keywords:  SI units; gentamicin; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR characterization; reference measurement procedure; traceability

DOI:  https://doi.org/10.1515/cclm-2026-0593
bioRxiv. 2026 Jul 28. pii: 2026.07.27.741082. [Epub ahead of print]

Optimizing Oxylipin Analysis with Liquid Chromatography Mass Spectrometry through Bio-Inert Systems and Ion Funnel Adjustments.

John T Shuster, Lingshuang Wu, Jericha Mill, Michaela Morhaus, Nina Yu Fan, Fernando Tobias, Dominique Baldwin, Matthew D Bruss, Liam Hurley, Michelle Kimple, Adam R Konopka, Judith Simcox.

Oxylipins are potent signaling lipids that affect inflammation, vascular tone, and metabolism, making them relevant in many diseases. Oxylipins are measured with liquid chromatography-mass spectrometry (LC-MS), but challenges in quantification arise due to low abundance and rapid degradation. In this study, we optimize LC-MS methods to improve the quantification of oxylipins in human plasma given growing interest in oxylipins and their impact on clinical research. Plasma samples were obtained from healthy participants and extracted by solid-phase extraction to concentrate the oxylipins. We then utilized a reversed phase targeted LC-MS/MS method using an Agilent 6495D triple quadrupole with transitions for 248 oxylipin species. Ion funnel voltages were set at 50 or 100 volts. Given the rapid degradation of oxylipins with bio-reactive surfaces, we compared both standard and Altura (bio-inert) columns, as well as standard and bio- inert LC setups. We observed that ion funnel parameters significantly alter detectable levels of oxylipins within LC-MS/MS analysis. By decreasing voltages applied to ions inside the ion funnel, signal was increased for most oxylipin species while peak quality was maintained. We also demonstrated that fully bio-inert setups quantify more compounds and show increased levels of some compounds, but fewer epoxyoctadecadienoic acid (EpODE) species. To explore this further, we injected analytical grade alpha-linolenic acid (ALA), the direct precursor of EpODEs, and observed formation of EpODEs within the instrumentation when using stainless steel columns. Our data shows that oxylipins benefit from fully bio-inert systems and optimized pre-mass analyzer parameters. The stainless-steel components of the column may also be contributing to epoxidation reactions of polyunsaturated fatty acids (PUFAs), generating oxylipin species during analysis. Finally, we utilized this method to perform oxylipin analysis in other human tissues including granulocytes, mononuclear cells, erythrocytes, skeletal muscle, and THP-1 cells, a human derived monocyte cell line.

DOI: https://doi.org/10.64898/2026.07.27.741082
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Aug 18. pii: S1570-0232(26)00348-X. [Epub ahead of print]1283 125259

Development and validation of an HPLC-MS/MS method for the quantification of Aficamten in human plasma and urine and application to a clinical pharmacokinetic study.

Xiaofei Wu, Aijing Liu, Adrienne Griffith, Hongzhong Liu, Xue Zhao, Hongyun Wang, Xiaohong Han.

  Aficamten (CK-3773274) is a next-in-class selective cardiac myosin inhibitor now approved for use for chronic oral therapy in hypertrophic cardiomyopathy (HCM). The present study established an innovative, rapid, and highly sensitive HPLC-MS/MS approach for quantifying aficamten concentrations in human plasma and urine. Samples were prepared using protein precipitation with acetonitrile containing CK-3942912 (D5-labelled aficamten) as the internal standard (IS). Sample separation was achieved using a C18 analytical column, with a mobile phase comprising acetonitrile and ammonium acetate. A Xevo TQS tandem mass spectrometer performed the detection in electrospray ionization (ESI) positive mode. The following MRM ion transitions were monitored: m/z 338.2 → 213.2 for CK-3773274 and m/z 343.2 → 218.2 for IS. This method was validated with excellent selectivity and sensitivity. Linear calibration curves (1-500 ng/mL) demonstrated excellent accuracy (RE% ±15%) and precision (RSD% ≤15%) for both intra- and inter-run analyses. Furthermore, critical validation parameters including matrix effects, extraction recovery, carryover, and stability were also assessed. The validated method was subsequently utilized to quantify aficamten concentrations in a clinical pharmacokinetic study involving healthy Chinese adults.

Keywords:  Aficamten; Bioanalysis; CK-3773274; HPLC–MS/MS; Human plasma and urine

DOI:  https://doi.org/10.1016/j.jchromb.2026.125259
Int J Cancer. 2026 Aug 19.

Spatially Resolved Metabolomics of Optimal Cutting Temperature (OCT) Compound-Embedded Tumors.

Joseph Monaghan, Nicholas Woytowich, Tian Zhao, Kiera Nguyen, Emily Mahony, Julian J Lum, Kyle D Duncan.

  Mass spectrometry imaging (MSI) is emerging as a powerful tool for uncovering the distribution of metabolites in the tumor microenvironment and studying tumor metabolism in vivo. To date, MSI of biobanked tissues contextualized by patient data has been limited to peptides, proteins, and glycans-with few examples for metabolites. This is because most biobanked fresh-frozen tissue required for spatial metabolomics is embedded in optimal cutting temperature (OCT) compound to preserve structural features and mitigate thermal decay. However, OCT introduces abundant polyethylene glycol and polyvinyl alcohol interferents. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to demonstrate MSI of metabolites in OCT-embedded tissue. Metabolite coverage and sensitivity for tissue mimetic homogenates embedded in OCT and an MSI-compatible material, carboxymethylcellulose (CMC), exhibited excellent agreement. We apply our ambient MSI workflow to study the impact of methionine-restriction in a preclinical mouse model undergoing adoptive T-cell therapy. After tumor incubation (8 days), lymphoma-bearing mice were maintained on a complete or methionine-restricted diet for 2 days. Nano-DESI MSI revealed a heterogeneous tumor microenvironment, with multiple methionine-cycle intermediates (S-adenosylmethionine, S-adenosylhomocysteine) and related metabolites, including known T-cell modulators (1-methylnicotinamide, polyamines) localizing to tumor subregions. Methionine-restricted tumors exhibited reduced methionine and elevated S-adenosylmethionine, relative to the control group. Overall, this work establishes the potential for spatial metabolomics of fresh-frozen OCT-embedded tumors, unlocking the wealth of information stored in primary tissue biobanks and consequently accelerating our understanding of cancer metabolism and treatment.

Keywords:  ambient ionization; mass spectrometry imaging; nanospray desorption electrospray ionization; spatial metabolomics; tissue preparation

DOI:  https://doi.org/10.1002/ijc.70707
Anal Bioanal Chem. 2026 Aug 19.

Decoding the urinary metabolome of aromatic amino acid pathways in Alport syndrome.

Domniki Gallou, Jaime Morillas-Armenta, Miguel Fernández-García, Belen Requena, Santiago Angulo, Olga Begou, Helen Gika, Georgios Theodoridis, Gema Fernández-Juárez, Amir Shabaka, Ana Gradillas, Coral Barbas, Víctor González-Ruiz, Alma Villaseñor.

  Chronic kidney disease (CKD) has been associated with alterations in plasma-free aromatic amino acids (AAA)-tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr)-and some downstream metabolites. However, AAA metabolism comprises a wider set of compounds, including microbiome-derived and sulfate-conjugated metabolites excreted in urine with potential biological implications. Alport syndrome (AS), a genetic condition defined by progressive renal impairment, frequently advances to CKD, suggesting that disturbances in AAA-related metabolism may also be relevant in this disorder. Nevertheless, a comprehensive quantitative method covering AAA-derived metabolites enabling an assessment of their implications in AS has not been established. Here, we introduce a novel method for the quantification of up to 43 AAA-derived metabolites in urine, including sulfated metabolites. The method is based on liquid chromatography coupled to tandem mass spectrometry and includes six deuterated internal standards to achieve reliable quantitation. A pooled healthy urine sample was characterized, and the method was validated in terms of linearity, matrix effect, accuracy, precision and sensitivity. The method was applied to urine samples from AS patients-with CKD and non-CKD-and controls. Significant alterations across groups in the concentrations of metabolites from Trp, Phe, and Tyr pathways were observed, including 4-OH-phenylacetic acid-O-sulfate. Notably, the urinary kynurenic acid (KYNA)/Trp ratio emerged as a potential indicator of renal function, showing a marked increase in AS patients with CKD, consistent with enhanced kynurenine pathway activation. Overall, this analytical platform represents a valuable tool for clinical research and for advancing the understanding of AAA-related metabolic dysregulation in renal diseases.

Keywords:  Aromatic amino acids; Kidney disease; MS/MS targeted metabolomics; Microbial metabolites; Sulfatome

DOI:  https://doi.org/10.1007/s00216-026-06707-8
Pharm Biol. 2026 Dec;64(1): 902-920

Simultaneous quantification of seventeen kratom alkaloids and metabolites in human plasma and its application to clinical pharmacokinetic sample analysis.

Siva Rama Raju Kanumuri, Manoj Gundeti, Sushobhan Mukhopadhyay, Naftali Z Zeilinger, C Austin Zamarripa, Kirsten E Smith, Christopher R McCurdy, Abhisheak Sharma.

   CONTEXT: Mitragyna speciosa Korth, commonly known as kratom, has been used traditionally in Southeast Asia by chewing fresh leaves and brewing decoctions (tea) for its stimulant and sedative-like properties. Despite its widespread use, controlled clinical trials are limited and have monitored mitragynine only or a few other major alkaloids and metabolites.
OBJECTIVE: The objective of the study was to develop and validate an ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of 12 kratom alkaloids along with 5 metabolites of mitragynine in human plasma.
MATERIALS AND METHODS: A sensitive UPLC-MS/MS based bioanalytical method for the simultaneous quantification of kratom alkaloids, including mitragynine, speciogynine, speciociliatine, mitraciliatine, paynantheine, isopaynantheine, corynantheidine, corynoxine-A, corynoxine-B, mitraphylline, speciofoline, and ajmalicine, along with five metabolites of mitragynine, 7-hydroxymitragynine, 9-hydroxycorynantheidine, mitragynine 16-carboxylic acid, mitragynine pseudoindoxyl, and 3-dehydromitragynine in human plasma was developed and validated following FDA guidelines. Chemically unstable metabolites, 7-hydroxymitragynine and 9-hydroxycorynantheidine, were stabilized in human plasma. The bioanalytical method was further used for the analysis of plasma samples collected during a clinical trial involving four regular kratom users.
RESULTS: The method was linear over the concentration range of 1-250 ng/mL. A simple protein precipitation method was used to extract analytes. Upon oral administration of kratom products, most of the alkaloids were absorbed quickly and showed peak plasma concentrations within 1-2 h post-dose.
DISCUSSION AND CONCLUSIONS: Mitragynine, speciociliatine, mitraciliatine, isopaynantheine, and paynantheine are major circulating alkaloids, with mitragynine 16-carboxylic acid and 9-hydroxycorynantheidine being the major circulating metabolites of mitragynine in plasma upon kratom oral administration.

Keywords:  Kratom; LC-MS/MS; clinical pharmacokinetics; metabolites; mitragynine

DOI:  https://doi.org/10.1080/13880209.2026.2715806
Angew Chem Int Ed Engl. 2026 Aug 19. e6117030

Resolving Spectral Complexity in 4D Lipidomics Using a Two-Dimensional Deconvolution Framework.

Yao Qian, Qirui Yu, Zhixu Ni, Zheng Ouyang, Xiaoxiao Ma.

  The application of data-independent acquisition (DIA) in 4D lipidomics has been constrained by spectral interference due to fragment ion overlap, a bottleneck that existing one-dimensional deconvolution methods fail to fully resolve. Here, we overcome this limitation by introducing a two-dimensional liquid chromatography-ion mobility (LC-IM) deconvolution framework that unlocks the full potential of 4D lipidomics. By mathematically modeling the orthogonal LC-IM separation dimensions, our method reconstructs high-quality MS/MS spectra from highly complex DIA data, effectively disentangling co-eluting lipid interferences. We demonstrate the power of this approach by annotating 491 lipids from 1 µL human plasma at a 1% false discovery rate, a two-fold increase in coverage compared to traditional methods. Beyond bulk analysis, we showcase its unique capability for spatial lipidomics, enabling deep profiling of laser-microdissected tissue regions equivalent to only hundreds of cells, revealing metabolic reprogramming in human hepatocellular carcinoma. We further integrate this workflow with six-plex isobaric labeling to achieve high-throughput, high-accuracy quantification in spatial tissue mapping. This transition from one- to two-dimensional deconvolution establishes a robust, sensitive platform for deep lipidome characterization, bridging the gap between proteomics-grade throughput and lipidomic structural complexity.

Keywords:  data‐independent acquisition; deconvolution; ion mobility; lipidomics; mass spectrometry

DOI:  https://doi.org/10.1002/anie.6117030
Methods Mol Biol. 2026 ;3055 169-178

Mass Spectrometry-Based Untargeted Metabolite Profiling to Discover Molecular Consequences of Mammalian Cell Infection with Fusobacterium nucleatum.

Qiuying Chen, Huiyong Cheng, Steven S Gross.

  Untargeted metabolite profiling, aka metabolomics, offers a powerful technology to potentially reveal fundamental molecular mechanisms whereby a perturbation in cell metabolism leads to an alteration in cell growth or physiological maintenance. Notably, this discovery approach does not require any preconceived notion of underlying molecular mechanisms, affording an opportunity to effectively shine a molecular mechanistic spotlight in a direction that an informed investigator may not have thought to consider. Fusobacterium nucleatum colonization has been linked to the initiation and promotion of the colorectal cancer and other pathologies. Although F. nucleatum infection can modulate mammalian cell signaling pathways involved in inflammation and host defense, the broad impact of F. nucleatum on host cell metabolism is poorly defined. Here, we describe the application of untargeted LC/MS-based profiling of cell metabolites as a tool to comprehensively assess the impact of F. nucleatum infection on mammalian cell metabolism. Untargeted metabolite profiling offers an unprecedented opportunity for the generation of novel molecular mechanistic hypotheses for subsequent confirmation by targeted orthologous investigations.

Keywords:  Fusobacterium nucleatum; Gram-negative bacteria; LC/MS; Liquid chromatography-mass spectrometry; Metabolomics; Spheroids; Untargeted metabolite profiling

DOI:  https://doi.org/10.1007/978-1-0716-5388-3_16
Anal Chim Acta. 2026 Oct 15. pii: S0003-2670(26)00915-3. [Epub ahead of print]1419 345965

New chromatographic and data evaluation approaches for the comprehensive investigation of metal-phytosiderophore-complex dynamics.

Laura Zellner, Andreea Spiridon, Sangeeta Kumari, Simone Braeuer, Nicolas Kratena, Christian Stanetty, Eva Oburger, Tim Causon, Christian W Klampfl, Stephan Hann.

   BACKGROUND: Phytosiderophores (PS) are root exudates released by graminaceous plants that play a crucial role in micronutrient acquisition. This is particularly relevant for iron, but PS can also complex other transition metals in the rhizosphere, influencing their bioavailability and uptake. However, characterizing intact metal-PS complexes remains analytically challenging, and studies have largely relied on targeted approaches using a limited number of selected PS.
RESULTS: We developed a liquid chromatography electrospray ionization high resolution mass spectrometry (LC-HRMS) workflow using a mixed-mode stationary phase to separate 2'-deoxymugineic acid, mugineic acid and 3″-epi-hydroxymugineic acid along with their Fe(III), Cu(II) and Ni(II) complexes. Complex integrity was preserved under mild gradient conditions, enabling quantification in negative ionization mode across a working range of 1 to 15 μmol L-1. In parallel, we utilized MetalPicker, a novel software tool for non-targeted detection of metal complexes in LC-HRMS data. By leveraging the distinctive isotopic distribution of transition metals, MetalPicker enables the discovery of metal-PS features in a non-targeted fashion. Validation of MetalPicker against a manufacturer software for targeted quantitative analysis yielded comparable results, demonstrating the accuracy and applicability of MetalPicker for non-targeted metal complex analysis. Our approach was applied for competitive complexation experiments in model samples, revealing a strong affinity of the PS towards Cu(II), potentially limiting iron bioavailability for plants.
SIGNIFICANCE: Our study presents the first chromatographic method separating metal-PS complexes alongside free PS, integrated with the novel non-targeted metal-complex detection software MetalPicker. Together, these advances enable the determination of coexisting metal-PS complexes in samples, expanding analytical capabilities for studying rhizosphere metal dynamics and nutrient mobilization.

Keywords:  LC-MS; Metal-complexes; Micronutrients; Phytosiderophores

DOI:  https://doi.org/10.1016/j.aca.2026.345965
Food Chem. 2026 Aug 19. pii: S0308-8146(26)03017-7. [Epub ahead of print]526 150857

Validation of an integrated analytical method for the simultaneous quantification of 12 PFAS in drinking water, plant-based milk alternatives and skimmed milk.

Bruna De Simone, Elvira De Rosa, Fabiana Di Duca, Paolo Montuori, Francesco Esposito, Teresa Cirillo.

  Per- and polyfluoroalkyl substances (PFAS) are persistent contaminants that may threaten food safety. Standardized analytical methods for low-fat liquid foods remain limited. This study validates a novel integrated workflow for the simultaneous determination of 12 PFAS in drinking water, plant-based milk alternatives and skimmed milk. Food samples underwent Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction, followed by one-step solid-phase extraction (SPE) cleanup for all matrices before ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis with isotope dilution. The method achieved excellent linearity (R2 ≥ 0.990), limits of quantification (LOQs) of 0.0005 μg/L for water and 0.0040 μg/kg for food matrices, and relative standard deviations (RSDs) < 20%. Isotopically labelled internal standards compensated matrix effects, avoiding matrix-matched calibration. Analysis of commercial samples showed PFAS concentrations below the LOQ in nearly all cases. This method provides a robust approach for multi-matrix PFAS monitoring and supports improved food safety surveillance.

Keywords:  Food safety; Isotope dilution; Matrix effects; Multi-matrix analysis; Per- and polyfluoroalkyl substances; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.foodchem.2026.150857
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Aug 15. pii: S1570-0232(26)00341-7. [Epub ahead of print]1282 125252

Determination of 13 per- and polyfluoroalkyl substances in human plasma samples using LC-MS/MS: application to capillary microsamples.

Julia Paim da Luz, Ana Julia Dossena, Eduarda Milena Reichert, Maine Lumertz Ferreira, Carolina Weber Ferrareze, Ana Paula Grando, Giovanna da Silva Grassi, Roberta Zilles Hahn, Mariele Feiffer Charão, Rafael Linden.

  Per- and polyfluoroalkyl substances (PFAS) are chemicals widely applied in industrial processes and highly persistent in the environment, whose extensive use has been linked to adverse health effects. Venous plasma is the conventional matrix for PFAS assessment in blood, and LC-MS/MS is the most used quantification technique. Despite the relevance of this topic, biomonitoring data on human exposure to PFAS in Brazil remain limited. This study validated an LC-MS/MS method for determination of 13 PFAS in human plasma. Blood samples were collected from volunteers by phlebotomy, followed by protein precipitation with acetonitrile containing 1% formic acid (v/v) and solid-phase extraction. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column. The assay was linear over a calibration range of 0.2-20 ng/mL. Intra- and inter-assay precision (CV%) were within the ranges of 2.06-12.0% and 0.25-10.7%, respectively. As for accuracy, results were 89.0-112.9%. Matrix effect ranged from -1.31 to 0.05%. Stability after four freeze/thaw cycles and under autosampler conditions were also confirmed for all analytes. The method was applied to 40 paired venous and capillary plasma samples. Both measures exhibited high correlation (r = 0.926). PFOS was the only compound detected at concentrations ≥0.2 ng/mL (LLOQ) in all samples, with capillary plasma concentrations of 0.85-13.50 ng/mL. In summary, the method showed good validation performance and demonstrated the suitability of capillary plasma samples as an alternative matrix for PFAS quantification.

Keywords:  Analytical validation; Capillary plasma; Human biomonitoring; LC-MS/MS; Per- and polyfluoroalkyl substances; Plasma

DOI:  https://doi.org/10.1016/j.jchromb.2026.125252
J Chromatogr A. 2026 Jul 21. pii: S0021-9673(26)00616-3. [Epub ahead of print]1786 467288

Development and validation of a multiplexed LC-HRMS method for nine GLP-1 receptor agonists and its pharmaceutical application.

Lok-Hang Tong, King-Kwong Leung, Chi-Tat Hung.

  A rapid, sensitive, and selective multiplexed liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed and validated for the identification and quantitation of nine structurally diverse peptide-based glucagon-like peptide-1 receptor agonists (GLP-1 RAs): bofanglutide, ecnoglutide, exenatide, liraglutide, mazdutide, retatrutide, semaglutide, survodutide, and tirzepatide. These peptides represent single, dual, and triple receptor agonists, including four globally approved medications, two recently approved by China's National Medical Products Administration (NMPA), and three investigational drugs in Phase III clinical trials. Chromatographic separation was achieved on an ACQUITY Premier BEH C18 column (2.1 × 150 mm, 1.7 µm) at 50 °C using a multistep linear gradient with water and acetonitrile containing 0.1% difluoroacetic acid (DFA) and 0.5% dimethyl sulfoxide (DMSO) as mobile phases (flow rate 0.3 mL/min). The optimised method provided excellent resolution with baseline separation for most peptides within a 20-minute runtime. A key advantage of this work is the implementation of a dual strategy that combines high-resolution full-scan MS (HRMS) for accurate mass measurement with data-dependent HRMS/MS fragmentation for structural confirmation. Peptide identification adopted a top-down approach, achieving mass accuracy of 0.0-0.5 ppm and fragment ion coverage of 76-100%. The method showed good linearity (r² ≥ 0.995) over 4-235 ng/mL, with LODs of 0.44-2.06 ng/mL and LOQs of 8.29-11.74 ng/mL. It was successfully applied to marketed formulations of liraglutide, semaglutide, and tirzepatide using a simple dilute-and-shoot procedure, yielding mean recoveries of 113.7-118.4%. This LC-HRMS method offers superior selectivity and flexibility for analysing current and next-generation GLP-1 RAs.

Keywords:  GLP-1 RAs; Glucagon-like peptide-1 receptor agonists; Multiplexed LC-HRMS; Peptide therapeutics

DOI:  https://doi.org/10.1016/j.chroma.2026.467288
PLoS One. 2026 ;21(8): e0356269

Targeted metabolomics of postmortem human cardiac tissue using the Biocrates MxP Quant 500 kit.

Oscar Emil Puntervold, Pia Johansson Heinsvig, Mads Gustav Skytte, Kristine Boisen Olsen, Jytte Banner, Jacob Tfelt-Hansen, Kristian Linnet, Marie Mardal.

AIM: This proof-of-concept study aimed to evaluate the feasibility and analytical performance of the Biocrates MxP® Quant 500 kit, originally developed for biofluids, to postmortem human cardiac tissue obtained from forensic autopsies, evaluating its potential as a standardized, cost-effective alternative to complex, resource-intensive metabolomics workflows.
METHODS: Left ventricular tissue samples were collected from 40 forensic autopsy cases, comprising 10 decedents with type 2 diabetes, 20 decedents with ischemic heart disease without type 2 diabetes, and 10 control cases without cardiac pathology. Cases were selected to represent the range of myocardial conditions commonly encountered in forensic practice, enabling assessment of analytical feasibility across heterogeneous postmortem cardiac tissue. Samples were analyzed using the MxP® Quant 500 kit following the standard protocol and using liquid chromatography-tandem mass spectrometry and flow injection analysis methods, measuring and quantifying a total of 630 endogenous metabolites across diverse classes.
RESULTS: Out of the 630 metabolites, 463 (74%) were within the quantifiable range. Lipid-related metabolites were notably well represented, with sphingomyelins (100% retained), phosphatidylcholines (93% retained), triacylglycerols (82% retained), and fatty acids (83% retained) showing the highest retention. Other metabolite classes such as acylcarnitines (45% retained) demonstrated greater variability, with some measurements falling below the limit of detection (e.g., 47% of acylcarnitines below this limit) or exceeding the upper limit of quantification (e.g., 35% of amino acids above this limit). Univariate analyses showed nominal group differences among specific metabolite subclasses (unadjusted p < 0.05). However, no metabolites remained statistically significant after correcting for false discovery rate. Multivariate analysis using PERMANOVA or PCA showed no strong global separation.
CONCLUSION: The Biocrates MxP® Quant 500 kit demonstrated technical feasibility for postmortem cardiac tissue analysis, enabling quantification of a broad range of metabolites, particularly lipids. While variability was observed across certain metabolite classes, the approach provides a promising basis for standardized metabolomic investigations in forensic and cardiovascular research.

DOI: https://doi.org/10.1371/journal.pone.0356269
Anal Methods. 2026 Aug 18.

Emerging analytical techniques for tamoxifen and metabolite quantification in diverse matrices.

Hemn A H Barzani, Rebaz Anwar Omer, Nergz Bayiz Abdulrahman, Hazha Omar Othman, Ali Sedeeq Noaman, Seerwan Hamadameen Sulaiman.

Tamoxifen (TMX), a key selective estrogen receptor (ER) modulator used in hormone-dependent breast cancer therapy, undergoes extensive metabolism to produce active metabolites, including endoxifen (END), 4-hydroxytamoxifen (4HT), and N-desmethyltamoxifen (NDT). The simultaneous determination of these compounds across pharmaceutical, biological, and environmental matrices remains analytically challenging due to their structural similarity, wide concentration range, strong protein binding, and significant matrix effects. This review critically evaluates emerging analytical techniques for TMX and its metabolites, focusing on chromatographic, electrochemical, and spectroscopic methods, and comparing their performance in terms of sensitivity, selectivity, reproducibility, cost, and matrix applicability. Among these, LC-MS/MS and UPLC-MS/MS methods demonstrate superior sensitivity (LOD as low as 0.03 ng mL-1) and high selectivity, making them well-suited for complex biological matrices and pharmacokinetic studies. In contrast, HPLC-UV and fluorescence methods are more applicable for pharmaceutical formulations due to their simplicity and cost-effectiveness, despite lower sensitivity. Electrochemical techniques offer rapid, low-cost analysis with enhanced sensitivity when using modified electrodes, although their selectivity and stability remain limited. Spectroscopic methods provide economical alternatives but are mainly restricted to high-concentration samples due to interference. Overall, method selection is highly matrix-dependent, and future developments should focus on improving sensitivity and selectivity, integrating green analytical chemistry, and advancing nanomaterial-based and AI-assisted analytical approaches.

DOI: https://doi.org/10.1039/d6ay01113f
Chem Commun (Camb). 2026 Aug 20.

Multi-omic analyses of the same sample using metabolomics, lipidomics, proteomics, phosphoproteomics, and glycoproteomics.

Yuefan Wang, Xuejun Peng, Hongyi Liu, Mia Gidley, Beth Kelly, Erika L Pearce, Edward J Pearce, Xianlin Han, Hui Zhang.

Mass spectrometry (MS)-based multi-omics offers powerful tools to comprehensively characterize proteins, post-translational modifications, metabolites, and lipids. However, these measurements are typically performed using separate sample preparation workflows and modality-specific liquid chromatography mass spectrometry (LC-MS) platforms, limiting integration and constraining applications to small amounts of sample materials, especially scarce clinical specimens. Here, we describe a unified nano-LC-MS framework that enables metabolomic, lipidomic, proteomic, phosphoproteomic, and glycoproteomic analyses from the same starting material using a single nano-LC-MS platform, with only the chromatographic conditions, acquisition methods, and enrichment procedures tailored to each omics. This integrated strategy reduces workflow complexity and sample consumption while improves analytical continuity across molecular layers. By enabling deep multi-omics characterization from the same sample, this platform provides a practical foundation for comprehensive analysis of precious clinical samples.

DOI: https://doi.org/10.1039/d6cc03477b
Methods Mol Biol. 2026 ;3035 91-104

Structure Analysis of Gangliosides: Mass Spectrometry Imaging.

Ariful Islam, Mitsutoshi Setou.

  Mass spectrometry imaging (MSI) is a powerful technique that is widely used for the mapping and molecular identification of biomolecules directly from the surface of biological samples. Among these biomolecules, gangliosides are complex glycosphingolipids primarily found in the nervous system. They play crucial roles in cell signaling and neurodevelopment and are responsible for many diseases. Label-free mapping and structural analysis of these gangliosides using MSI offer unique insights into their spatial distribution and concentration in healthy and diseased tissues. MSI techniques such as matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) have been widely employed to study gangliosides. These MSI tools enable high-resolution visualization of specific ganglioside species, contributing to the understanding of their roles in human diseases including neurodegenerative diseases, cancer, etc. Continuous improvements in sensitivity and resolution of MSI tools, continues to offer new insights into ganglioside biology, paving the way for novel therapeutic approaches in a wide range of diseases. Here, we have described the mapping and structural analysis of gangliosides using MSI.

Keywords:  DESI; Gangliosides; MALDI; Mass spectrometry imaging; Neurodegenerative diseases

DOI:  https://doi.org/10.1007/978-1-0716-5288-6_8
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Aug 20. pii: S1570-0232(26)00349-1. [Epub ahead of print]1283 125260

Analytical strategies for complex injectables: the expanding role of LC-MS technologies.

Niraj Shukla, Sunil Pawar, Mamta Pandey.

  Complex injectable therapeutics, including lipid nanoparticles, antibody-drug conjugates, biotherapeutics, and long-acting delivery systems, present analytical challenges that frequently exceed the capabilities of conventional pharmaceutical testing approaches. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a key analytical platform for addressing these challenges through sensitive quantification, structural characterization, and quality assessment of complex drug products. This review critically examines the evolving role of LC-MS in the development, characterization, quality evaluation, and regulatory assessment of complex injectable formulations, with emphasis on the analytical considerations that influence method selection and performance. Particular attention is given to the capabilities and limitations of contemporary LC-MS approaches for increasingly heterogeneous and structurally complex formulations. Despite significant advances, challenges associated with matrix complexity, data interpretation, and analytical harmonization continue to limit broader implementation. Future progress will depend on standardized analytical workflows, improved reference materials, and greater integration of advanced mass spectrometry technologies into pharmaceutical development and quality systems. Overall, LC-MS has evolved into an indispensable platform for ensuring the quality, safety, and performance of next-generation complex injectable therapeutics.

Keywords:  Bioanalysis; Complex injectables; Free drug content; In-vitro release; LC-MS; Matrix effects; Structural characterization

DOI:  https://doi.org/10.1016/j.jchromb.2026.125260
Talanta. 2026 Aug 08. pii: S0039-9140(26)01082-9. [Epub ahead of print]312(Pt B): 130426

A systematically evaluated SEC-ICP-MS/MS workflow for Mn, Fe, Cu, and Zn speciation in human serum.

Johannes Fischer, Anne Nagel, Andy Schmied.

  Species-resolved analysis of essential transition metals in serum may provide biologically and clinically relevant information beyond total elemental concentrations, particularly where altered metal distribution is linked to toxicological or disease-related processes. However, serum metal speciation remains analytically challenging because many metal-protein and metal-ligand interactions are labile, relevant fractions span broad molecular-mass and concentration ranges, and species-specific standards are generally unavailable. This work presents a newly developed size exclusion chromatography-inductively coupled plasma tandem mass spectrometry (SEC-ICP-MS/MS) workflow for combined Mn, Fe, Cu, and Zn speciation in human serum. It integrates bioinert chromatography, mild mobile-phase conditions, and matrix-matched calibration across the chromatographic system. Importantly, its analytical performance was systematically assessed over five measurement days using two quality control materials and two native human serum samples, addressing an aspect that has received limited attention in previous multi-element serum speciation studies. The summed chromatographic response showed excellent linearity, and mean total-element recoveries after chromatographic separation were 96-102 %. Major species exhibited intra-day coefficients of variation (CVs) below 1.5 % and inter-day CVs below 5 %, while remaining fractions stayed below 10 % and 15 %, respectively. Limits of quantification were sufficiently low for all investigated elements, including low-abundance Mn. The method reproducibly resolved four Mn, three Fe, five Cu, and four Zn species, most of which could be tentatively assigned to biologically plausible proteins. Overall, the method provides a systematically evaluated multi-element platform that may help move serum metal speciation beyond predominantly exploratory applications toward more demanding biomonitoring, occupational, and clinical study designs.

Keywords:  Human serum; Metalloproteins; SEC-ICP-MS/MS; Serum manganese; Serum metal speciation; Size exclusion chromatography; Trace element speciation

DOI:  https://doi.org/10.1016/j.talanta.2026.130426
Talanta. 2026 Aug 17. pii: S0039-9140(26)01120-3. [Epub ahead of print]312(Pt B): 130464

Development and validation of a cost-effective and reliable HPLC-FLD method for cortisol determination in human hair.

Mohamed A Gab-Allah, Hyojin Hwang, Maryam Adelipour, Kyu H Park, Jeongkwon Kim.

  Hair analysis is increasingly employed to assess long-term exposure to endogenous and exogenous substances, particularly steroid hormones. This study presents a simple, robust, and cost-effective high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for the determination of cortisol in human hair. Cortisol was extracted with methanol and derivatized using a sulfuric acid/ethanol (70:30, v/v) reagent at 70 °C. The sample extract was then neutralized with 1 M ammonium bicarbonate to eliminate residual acidity and purified by solid-phase extraction (SPE) cartridges. Two different SPE cartridges, including Oasis HLB and Bond Elut C18, were comparatively evaluated with respect to cortisol recovery and matrix clean-up efficiency. Derivatization parameters and chromatographic conditions were systematically optimized to maximize analytical performance. The method was validated according to ICH M10 bioanalytical guidelines over a concentration range of 5-200 pg/mg. Matrix-matched calibration exhibited excellent linearity (R2 > 0.999), with limits of detection and quantification of 1.1 and 3.5 pg/mg, respectively. Average recoveries ranged from 89% to 108%, while intra- and inter-day precision remained below 6% and 9%, respectively. Matrix effects were minimal (15.1%), and greenness assessment using four complementary metrics confirmed the environmentally favorable characteristics of the method. Overall, the proposed HPLC-FLD method provides a practical and accessible alternative to LC-MS/MS for routine hair cortisol analysis, combining simplified sample preparation, adequate sensitivity, reduced solvent consumption, and lower operational costs.

Keywords:  Cortisol; Derivatization; HPLC-FLD; Human hair; Method validation

DOI:  https://doi.org/10.1016/j.talanta.2026.130464
Food Res Int. 2026 Oct 31. pii: S0963-9969(26)01557-7. [Epub ahead of print]242(Pt 1): 119873

Mass spectrometry similarity analysis and pattern recognition-driven metabolomics strategy for species and geographic chemotyping of Gardenia fruit.

Yuan Mao, Qi Huang, Liwen Huang, Fenghua Kang, Hongping Long, Zhaoquan Wu, Yikun Wang.

  Gardenia fruit possesses significant medicinal and edible value; however, its quality control remains challenging due to adulterant confusion and geographical diversity. Herein, an integrated mass spectrometry (MS1)/MS2 similarity filtering strategy including polygonal mass defect filtering, diagnostic ion and neutral loss analysis, and feature-based molecular networking was established to profile diverse chemicals of Gardenia fruit, preliminarily screening 3675/1942 flavonoids, 5642/2329 phenylpropanoids, 2502/1061 iridoids, 937/250 non-iridoid monoterpenes, 825/486 diterpenes, and 76/33 triterpene aglycones in ESI+/ESI- mode, and finally identifying 5 potentially new phenylpropanoid derivatives. Moreover, multivariate statistics and machine learning informed UPLC-Q-TOF-MS/MS-based untargeted metabolomics analysis were applied to compare metabolic differences and screen potential markers for their species authentication and geographic chemotyping. The key species marker set composed of d-mannitol, fumaric acid and asiatic acid generated a binary classifier with 100% accuracy that discriminated Gardenia fruit from its adulterants according to their characteristic abundance. And key geographic chemotyping markers were screened as genipin 1-gentiobioside combined with jasminoside B, a binary classifier built on their abundance also achieved 100% accuracy. This study offers robust technical and methodological support for the quality assessment, authentication and geographic chemotyping of Gardenia fruit.

Keywords:  Gardenia fruit; Geographic chemotyping; Mass spectrometry similarity analysis; Quality assessment; Species authentication; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.foodres.2026.119873
Crit Rev Anal Chem. 2026 Aug 17. 1-50

Mass Spectrometry-Based Anti-Doping Analysis: A Critical Review of Emerging Analytical and Sample Pretreatment Strategies.

Dnyaneshwar Maykar, Pratik Vilas Gore, Suraj R Chaudhari, Pramod Kumar.

  Illicit use of doping substances has raised significant ethical and health concerns in sports. Therefore, continuous doping control is essential for maintaining fairness and athlete's safety. Accurate and rapid detection of prohibited substances at their lowest levels in complex biological matrices, such as blood, urine, and plasma, is crucial for reliable anti-doping analysis. This critical review focuses on the applications of mass spectrometry (MS) for the detection of doping agents across diverse biological matrices. Special emphasis is given to advanced mass spectrometry platforms, particularly LC-QQQ-MS, LC-QTOF-MS, Orbitrap-MS/MS, and IRMS which are widely used for the routine screening, confirmation, and identification of emerging doping agents. Microsampling approaches such as dried blood spots (DBS) and volumetric absorptive microsampling (VAMS) provide superior alternatives to traditional sampling. Furthermore, comprehensive understanding of sample pretreatment and analytical techniques is essential. In this regard, novel microextraction techniques, including solid-phase microextraction (SPME), liquid-phase microextraction (LPME), supramolecular solvents (SUPRAS), and deep eutectic solvents (DES), have improved multi-analyte sample pretreatment strategies and facilitated advanced automation. This review also offers valuable guidance in selecting appropriate chromatography coupled mass spectrometry‑based analytical methods with sensitive detection, sample pretreatment techniques, and storage conditions during sports drug testing.

Keywords:  Automation; LC-MS/MS; doping agents; microextraction; microsampling

DOI:  https://doi.org/10.1080/10408347.2026.2715148
Rapid Commun Mass Spectrom. 2026 Nov 15. 40(21): e70168

Automated Tissue Classification and Candidate Biomarker Feature Extraction in Mass Spectrometry Imaging Based on Interpretable Deep Learning Algorithm.

Guang Xu, Shengfeng Gan, Bo Guo, Li Yang.

   RATIONALE: Mass spectrometry imaging (MSI) generates high-dimensional spatial-spectral data that requires efficient computational methods for tissue classification and candidate biomarker feature extraction. Deep learning offers a promising approach, yet the interpretability of model predictions and identification of biologically relevant spectral features remain challenging.
METHODS: A comprehensive computational pipeline was developed for automatic tissue layer classification of a public mouse urinary bladder MSI dataset. Building upon prior work that compared manual tissue layer labels with those automatically generated via spectral preprocessing, t-SNE, and hierarchical clustering, in this study we separately use each type of class label to train convolutional neural networks (CNNs) for supervised classification. Gradient-weighted Class Activation Mapping (Grad-CAM) and SHapley Additive exPlanations (SHAP) were employed to compute layer-specific summed importance scores to evaluate each mass spectral feature and extract class-discriminative features.
RESULTS: On the mouse urinary bladder MSI dataset, both manual labels and cluster-derived labels (t-SNE + hierarchical clustering) enabled the CNN model to achieve training accuracies exceeding 0.9 for classifying three tissue layers. Interpretability methods successfully identified discriminative m/z features, including known lipids such as SM(34:1) (m/z 741.54) and PC(34:1) (m/z 798.54), consistent with previously reported biological markers. Compared to the intensity values, the importance scores of the top class-discriminative features generated by both interpretability methods exhibited a sharper contrast and superior ability to delineate tissue-layer-specific distributions in their ion images. Furthermore, evaluation on an independent colorectal cancer dataset yielded a test accuracy of 0.758, suggesting that cross-patient generalizability varied across different data sources.
CONCLUSIONS: This study presented an effective deep learning framework for accurate and interpretable tissue classification in MSI data. The CNN modeling and deep learning interpretability provided a robust approach for both automated segmentation and biological discovery, facilitating the identification of spatially resolved metabolic features in tissue sections.

Keywords:  CNN; Grad‐CAM; MSI; SHAP; biomarker feature; deep learning

DOI:  https://doi.org/10.1002/rcm.70168
J Cheminform. 2026 Jul 31. pii: 114. [Epub ahead of print]18(1):

How well can off-the-shelf LLMs elucidate molecular structures from mass spectra using chain-of-thought reasoning?

Yufeng Wang, Lu Wei, Lin Liu, Hao Xu, Haibin Ling.

  Mass spectrometry (MS) is a powerful analytical technique for identifying small molecules, yet determining complete molecular structures directly from tandem mass spectra (MS/MS) remains a long-standing challenge due to complex fragmentation patterns and the vast diversity of chemical space. Recent progress in large language models (LLMs) has shown promise for reasoning-intensive scientific tasks, but their capability for chemical interpretation is still unclear. We present a zero-shot chain-of-thought (CoT) benchmark for evaluating how well off-the-shelf general-purpose LLMs can infer molecular structure from text-serialized MS/MS peak lists and a known molecular formula, without fine-tuning, retrieval, external chemistry tools, or constrained search. Rather than proposing a new spectrum-trained model, our goal is diagnostic: to isolate what the evaluated off-the-shelf LLMs can and cannot do in a direct MS/MS-to-SMILES generation setting when only textualized spectral evidence and prompting are available. This distinction is important because our benchmark targets a deliberately constrained regime: direct structure generation from text-formatted spectral evidence alone, rather than multimodal reasoning with spectral images, candidate reranking over structures generated by specialized chemistry systems, or knowledge and search-augmented LLM workflows. We formalize expert chemists' reasoning steps (such as double bond equivalent (DBE) analysis, neutral loss identification, and fragment assembly) into structured prompts and assess multiple state-of-the-art LLMs (Claude-3.5-Sonnet, GPT-4o-mini, and Llama-3 series) using the MassSpecGym dataset. Our evaluation across metrics of SMILES validity, formula consistency, and structural similarity reveals that while LLMs can produce syntactically valid and partially plausible structures, they fail to achieve chemical accuracy or link reasoning to correct molecular predictions. These findings highlight both the interpretive potential and the current limitations of LLM-based reasoning for molecular elucidation, providing a foundation for future work that combines domain knowledge and reinforcement learning to achieve chemically grounded AI reasoning.Scientific contributionWe introduce a zero-shot chain-of-thought benchmark for direct MS/MS-to-SMILES prediction with off-the-shelf general-purpose LLMs, providing a controlled test of text-only, no-tools molecular structure elucidation. In contrast to prior approaches based on spectrum-trained models, multimodal reasoning, or tool-augmented candidate pipelines, our study isolates the capabilities and limitations of prompting alone. We show that fluent chain-of-thought reasoning does not translate into chemically grounded structure recovery, establishing a reproducible baseline and a clear target for future method development.

Keywords:  Chain-of-thought; LLM; Mass spectra; Molecular structures

DOI:  https://doi.org/10.1186/s13321-026-01234-1
J Pharm Anal. 2026 Aug;16(8): 101596

Integrating mass spectrometry imaging and data-driven segmentation for spatial metabolic mapping of diabetic eye disease.

Shuohan Cheng, Shuo Wang, Tianfang Lan, Hongtao Jin, Zhi Zhou, Zhonghua Wang, Zeper Abliz.

  Diabetic eye disease (DED) is a leading cause of vision impairment worldwide, yet the molecular mechanisms underlying its progression remain incompletely understood. In this study, we applied a dual-platform spatial metabolomics strategy integrating air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) and matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to characterize spatial metabolic alterations in the eyes of diabetic rats. Data-driven segmentation of retinal micro-regions using SCiLS Lab software enabled fine-scale mapping of metabolic heterogeneity. Physiological, biochemical, and histopathological analyses were combined with spatial metabolite mapping to construct a metabolic atlas and evaluate the regulatory effects of ferulic acid. We established a comprehensive spatial metabolome atlas of the rat eye, identifying 135 annotated metabolites and revealing significant region-specific metabolic heterogeneity. Unsupervised k-means clustering was further applied to the high-resolution MALDI-MSI data, successfully delineating distinct functional micro-regions of the retina solely based on endogenous metabolic profiles, demonstrating the power of data-driven tissue segmentation. In diabetic eyes, 39 metabolites were significantly dysregulated, involving amino acid, glucose, lipid, and redox metabolism. Notably, lysine, arginine, carnitine, and glutathione (GSH) were depleted, while glucose-6-phosphate (G6P), glycerol-3-phosphate (G3P), and pro-inflammatory lipids were elevated, highlighting profound metabolic reprogramming across ocular compartments. Ferulic acid treatment restored nine key metabolites, alleviated oxidative stress, normalized lipid and glucose metabolism, and improved retinal structural integrity in a dose-dependent manner. This study shows that integrating mass spectrometry imaging with data-driven tissue segmentation reveals spatial metabolic reprogramming in DED and highlights ferulic acid as a promising therapeutic candidate.

Keywords:  Air-flow-assisted desorption electrospray ionization; Diabetic eye disease; Ferulic acid; Mass spectrometry imaging; Matrix-assisted laser desorption ionization; Metabolic reprogramming

DOI:  https://doi.org/10.1016/j.jpha.2026.101596
Anal Chem. 2026 Aug 18. 98(32): 23920-23928

Metal-Anchored Silicon-Based Nanostructures Enable Dual-Polarity SALDI-MS Analysis of Small Molecules.

Kenan Chen, Yanan Wang, Siying Fang, Xinchun Du, Yiyi Ye, Qinyu Jiang, Keren Cheng, Xuetong Qu, Jianmin Wu.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is an attractive technique for small molecule analysis, because of its low background interference and high throughput. However, the performance of SALDI-MS is strongly dependent on substrate design, and the simultaneous realization of dual-polarity ionization, high reproducibility, and high-throughput capability remains challenging. Herein, we report an ordered silicon-based nanostructure array chip anchored with gold nanoparticles (VSiNW-Au) as an efficient SALDI-MS substrate for small-molecule analysis. The uniform anchoring of gold nanoparticles on silicon nanowire arrays affords low background interference, enhanced ionization efficiency, and improved reproducibility, while supporting dual-polarity SALDI-MS analysis under both positive and negative ion modes. The hydrophobic array architecture, combined with automated mass spectrometry data acquisition, enables rapid analysis of up to 60 samples within 10 min. The performance of the VSiNW-Au platform is demonstrated by detecting perfluoroalkyl and polyfluoroalkyl substances (PFAS) in spiked water samples and by comprehensive profiling of metabolites and lipids in serum. These results highlight the potential of the VSiNW-Au array chip as a robust and high-throughput SALDI-MS platform for rapid screening applications in environmental and biological analysis.

DOI: https://doi.org/10.1021/acs.analchem.6c04358
Drug Test Anal. 2026 Aug 19.

Development and Validation of an HPLC-MS/MS Method for the Determination of Benzodiazepines in Hair. Comparison of Two Different Extractions and Applications on Real Samples.

Martina Pes, Giulia Stocchero, Manuela Lucchiari, Antonio Cambria, Giovanni Michele Marchio, Flora Donini, Francesco Ingenito, Lucia Bertoldi, Lucia Pecoraro, Alberto Salomone, Donata Favretto, Adriano Anesi.

  Benzodiazepines are widely prescribed psychoactive drugs frequently encountered in forensic and clinical toxicology. Hair analysis is a useful tool for retrospective drug monitoring, but its effectiveness depends on highly sensitive and reliable analytical methods due to the complexity of the matrix and the typically low concentrations of analytes, especially after single-dose exposure. This study aimed to develop, validate, and compare two LC-MS/MS methods for the simultaneous identification and quantification of 38 benzodiazepines and their metabolites in 25 mg of hair. Both methods used identical procedures for sampling, washing, and instrumental analysis, differing only in the extraction step. In the first method, 500 μL of M3 solution was added to cut hair and incubated at 100°C for 60 min. In the second, methanol was added to pulverized hair and incubated overnight at 50°C. Extracts were evaporated, reconstituted in 500 μL of M3, and 3 μL injected into the LC-MS/MS system. Validation with spiked hair and quality control samples showed limits of quantification ranging from 2 to 46 pg/mg, with linear calibration curves up to 200 pg/mg. Imprecision was ≤ 20% (n = 9), and most analytes showed process efficiencies above 70%. Application to 72 real samples demonstrated that the M3 extraction provided lower detection limits than the methanol method, making it more suitable for identifying single-dose exposure. Both methods are suitable for benzodiazepine screening in forensic toxicology, particularly when sample amounts are limited.

Keywords:  HPLC‐MS/MS; benzodiazepines; cut hair; powdered hair; validation

DOI:  https://doi.org/10.1002/dta.70142
Biomed Chromatogr. 2026 Oct;40(10): e70565

Quantitation of Cirtuvivint (SM08502) in Human Plasma by LC-MS/MS.

Julianne L Holleran, Joshua J Deppas, Ye Feng, Michael J Hochman, Evan C Chen, Maximilian Stahl, Steven D Gore, Timothy W Synold, Jan H Beumer, Raman Venkataramanan, Robert A Parise.

  CDC-like kinases (CLK) and dual-specificity tyrosine-regulated kinases (DYRK) are protein kinases involved in various cellular functions, mRNA splicing, and DNA damage repair. CLK/DYRK kinases have been implicated in many disorders such as diabetes, neurodegenerative diseases, and cancer. Cirtuvivint (SM08502) is an orally bioavailable, first-in-class pan-CLK and pan-DYRK inhibitor that modulates pre-mRNA splicing and has shown the ability to inhibit cancer cell growth in vitro and reduce tumor burden in vivo. This has led to the administration of cirtuvivint in phase I clinical trials. To quantitate cirtuvivint, we have developed and validated an LC-MS/MS method in human plasma. The assay is simple and robust and consists of a protein precipitation, dilute-and-shoot extraction method using 20 μL of plasma, chromatographic separation with a Phenomenex Kinetex C18, and a gradient mobile phase system consisting of 0.1% formic acid in water and acetonitrile. The chromatographic method is followed by mass spectrometric detection with a SCIEX 4500 tandem mass spectrometer. The method has a 5-min run time and is linear from 5 to 1000 ng/mL. The assay met the criteria outlined by the US Food and Drug Administration guidance for bioanalytical method validation and will support ongoing and future clinical studies defining cirtuvivint pharmacokinetics.

Keywords:  LC–MS/MS; SM08505; cirtuvivint; human plasma; pharmacokinetics; validation

DOI:  https://doi.org/10.1002/bmc.70565