bims-metlip 2026-10-11 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–10–11
23 papers selected by
Sofia Costa, Matterworks

LC-MS/MS quantification of N-lactoyl-phenylalanine and N-lactoyl-tyrosine in human plasma: method validation and clinical application to metformin administration.
Correlating metabolites, lipids, and N-glycans in murine kidney microstructures via integrative mass spectrometry imaging.
Unlocking the bile acid universe: advanced workflows and a multidimensional library of 280 unique species.
Determination of 5-Fluorouracil, 5,6-dihydro-5-Fluorouracil, Uracil and Dihydrouracil by UPLC-MS/MS in plasma and its application in hepatocellular carcinoma.
Multi-reflecting time-of-flight mass spectrometry enables high-accuracy metabolite identification in a demonstration study using serum samples from COVID-19 patients.
Metabonaut: an open educational resource for learning reproducible, script-based untargeted LC-MS/MS metabolomics.
Simultaneous Quantification of Vedolizumab, Ustekinumab, and Infliximab in Human Serum by LC-MS/MS: Application to Therapeutic Drug Monitoring in Inflammatory Bowel Disease.
LC-MS/MS method for quantification of dupilumab in human serum using Protein G enrichment.
A Comprehensive Dual Platform, End-to-end Workflow for Metabolic Profiling of Dried Blood Microsamples.
From Single Injection to Dual Insights: Integrating EAD and CID Acquisition for Spectral Library Generation and EAD-CID Spectral Matching.
Targeted quantification of 25 tryptophan metabolites across three metabolic pathways in serum by ultra-high-performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry.
Evaluation of a Hydrophilic Interaction Liquid Chromatography Design Space for Amino Acids.
Metabolite-targeted LC-HRMS quantification of emerging indazole-based synthetic cannabinoids in urine samples from an addiction treatment cohort.
Nanomatrix engineering for LDI-MS based biomedical metabolomics.
Orthogonality-driven offline two-dimensional liquid chromatography coupled with high-resolution mass spectrometry for comprehensive chemical profiling and quality marker screening of Renshen Guben oral liquid.
Recent Advances in Sample Preparation for Cholesterol and Oxysterol Analysis in Complex Biological Matrices.
Lipid profiling of meat samples using surface extraction followed by multiple reaction monitoring profiling.
Integrated assessment of fatty acid metabolism and cellular energy status using HILIC-MS/MS.
Phenolic-focused untargeted ion mobility LC-MS metabolomics reveals season-associated chemical signatures in Scottish honey.
Comprehensive evaluation of metabolite imputation methods and the effect of imputation accuracy on the performance of metabolite-trait association using large-scale metabolite datasets in cattle.
Universal charge-tag labelling of undetectable polymers for mass spectrometric characterization.
Green Sorptive Sampling Coupled With UHPLC-IMS-MS and GC×GC-TOFMS for Characterization of Migrating Substances From Plastic Food Contact Materials.
Analytical performance and clinical classification value of a novel sandwich immunoassay for total testosterone measurement in low-concentration and hemodialysis populations.

Transl Clin Pharmacol. 2026 Sep;34(3): 172-181

LC-MS/MS quantification of N-lactoyl-phenylalanine and N-lactoyl-tyrosine in human plasma: method validation and clinical application to metformin administration.

Ga-Yeong Lee, Yeonseo Jang, Jae-Yong Chung, Joo-Youn Cho.

  N-lactoyl amino acids, including N-lactoyl-phenylalanine (Lac-Phe) and N-lactoyl-tyrosine (Lac-Tyr), have recently attracted interest as endogenous metabolites of potential clinical and metabolic relevance. In particular, Lac-Phe has been implicated in appetite regulation and in the metabolic effects of metformin. However, validated analytical methods for the simultaneous quantification of these endogenous metabolites in human plasma remain limited. In this study, a sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of Lac-Phe and Lac-Tyr in human plasma using a simple protein precipitation procedure with a 50-μL sample volume. To minimize interference from endogenous analytes, an eight-point calibration curve was prepared using an artificial surrogate matrix. The assay showed a calibration range of 0.5-100 ng/mL for both analytes, with a lower limit of quantification of 0.5 ng/mL and a total run time of 5.5 minutes. The method demonstrated acceptable analytical performance for both Lac-Phe and Lac-Tyr, with intra- and inter-day precision values of ≤ 8.8% and mean accuracy ranging from 88.0% to 101.4%. The validated assay was successfully applied to plasma samples from healthy volunteers collected before and after metformin administration. This method provides a reliable analytical tool for the simultaneous quantification of Lac-Phe and Lac-Tyr in human plasma and may facilitate future clinical and pharmacological investigations of lactoyl-amino acid metabolism.
Trial Registration: ClinicalTrials.gov Identifier: NCT04335526.

Keywords:  Chromatography, Liquid; Metformin; N-Lactoyl-Phenylalanine; Tandem Mass Spectrometry

DOI:  https://doi.org/10.12793/tcp.2026.34.e12
Nat Commun. 2026 09 04. pii: 10544. [Epub ahead of print]17(1):

Correlating metabolites, lipids, and N-glycans in murine kidney microstructures via integrative mass spectrometry imaging.

Nina Ogrinc, Rick Ursem, Hans Dalebout, Jesper Kers, Martin Giera, Manfred Wuhrer, Noortje de Haan.

Understanding how metabolites, lipids and glycans interact within tissues at the same cellular layer is key to uncovering their roles in health and disease. Herein, we present an integrative multi-omic mass spectrometry imaging (MSI) approach correlating metabolites, lipids and N-glycans, including their linkage-specific sialic acid information, in a single renal tissue section. Our protocol enables the sequential detection of hundreds of metabolites and lipids species alongside diverse glycosylation traits in small cell clusters across the kidney. Importantly, our pixel-to-pixel co-registration and clustering correlates glycans (recorded in positive mode MS), metabolites, and lipids (both in negative mode MS) across kidney microstructures. This analysis reveals strong association between taurine, TCA metabolites, sulfatides, and polyunsaturated phospholipids, and specific sialic acid linkages on N-glycans in the thick ascending limb and collecting ducts. The robust strategy maximizes molecular insights from scarcely available samples and offers a powerful tool for exploring tissue complexity in both normal and disease states.

DOI: https://doi.org/10.1038/s41467-026-77127-6
Sustain Microbiol. 2026 ;3(4): qvag039

Unlocking the bile acid universe: advanced workflows and a multidimensional library of 280 unique species.

Guozhi Zhang, Emily C Vincent, Sadie M Disselkoen, James N Dodds, Quentin DuVal-Smith, Abubaker Patan, Ipsita Mohanty, Victoria Deleray, Jian Zhang, Paul A Thiessen, Evan E Bolton, Emma L Schymanski, Pieter C Dorrestein, Casey M Theriot, Erin S Baker.

  Microbes and bile acids are tightly intertwined, especially in the gut. While the liver produces primary bile acids from cholesterol, gut bacteria transform these into diverse secondary forms which act as powerful signaling molecules. Since bile acid changes are increasingly linked to health and disease, their accurate measurement in the gut and circulation is essential. Bile acid analytical evaluations, however, are challenging as many co-elute in liquid chromatography (LC), share identical mass spectrometry (MS) precursor masses, and produce similar tandem mass spectrometry (MS/MS) spectra. As a result, conventional LC-MS/MS workflows struggle to differentiate bile acids and have motivated the addition of orthogonal separations such as ion mobility spectrometry (IMS). Here, we assess optimal bile acid extraction parameters for stool, serum, and plasma; compare LC conditions; and assess electrospray ionization performance across polarities. Additionally, we introduce a publicly available multidimensional reference library containing LC retention times, IMS collision cross section values, and accurate precursor masses for 280 unique bile acids (264 endogenous and 16 deuterium-labeled species) including unconjugated, host-conjugated, and microbially conjugated bile acids. This multidimensional library empowers bile acid identification in complex samples and enables a more comprehensive exploration of their biological roles and disease associations.

Keywords:  bile acid extraction; bile acids; collision cross section; ion mobility spectrometry; suspect screening

DOI:  https://doi.org/10.1093/sumbio/qvag039
Anal Biochem. 2026 Oct 06. pii: S0003-2697(26)00223-X. [Epub ahead of print]720 116267

Determination of 5-Fluorouracil, 5,6-dihydro-5-Fluorouracil, Uracil and Dihydrouracil by UPLC-MS/MS in plasma and its application in hepatocellular carcinoma.

Zhiqiang Tong, Tingting Ding, Yuexin Li, Dongqing Gao, Zhonghua Sun, Xuemei Chen, Shuai Jiang, Mei Dong.

   OBJECTIVE: Aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 5-Fluorouracil (5-FU), 5,6-dihydro-5-Fluorouracil (5-FUH2), Uracil (U), and Dihydrouracil (UH2) concentrations in human plasma, and to apply 5-FU therapeutic drug monitoring(TDM).
METHODS: UPLC-MS/MS method was adopted using 5-bromouracil (5-Bru) as internal standard (IS).The determination was performed on Waters Acquity UPLC BEH Amide column (100 mm × 2.1 mm,1.7 μm) with mobile phase consisted of acetonitrile and water. An electrospray ion source was used to carry out negative ion scanning with multiple reaction monitoring (MRM).The method was applied to 10 patients receiving FOLFOX-HAIC. 5-FU exposure was estimated as AUC = Css × TCI, and UH2/U ratio was explored as a DPD-related metabolic marker.
RESULTS: Accuracy, precision, recovery, matrix effect, and stability were examined to meet ICH M10 guidelines for validation of biological methods.All four analytes were quantified over 20-1000 ng/mL with a lower limit of quantification of 20 ng/mL. Mean extraction recovery was approximately 52%-58%, with matrix effects close to 100%.Among 10 patients with advanced hepatocellular carcinoma, the Css of 5-FU ranged from 202.39 to 583.70 ng/mL, the AUC of 5-FU ranged from 9.31 to 26.85 mg h/L, and the plasma concentration of 5-FUH2 ranged from 352.96 to 892.59 ng/mL. The UH2:U concentration ratio ranged from 0.49 to 7.83.
CONCLUSION: We developed a rapid 5-min UPLC-MS/MS method for simultaneously determining 5-FU, 5-FUH2, U and UH2 in human plasma and applied it to patients treated with FOLFOX-HAIC. This method can comprehensively evaluate the 5-FU exposure and pyrimidine metabolism of patients with hepatocellular carcinoma.

Keywords:  5-FU; HAIC; TDM; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.ab.2026.116267
Metabolomics. 2026 Oct 03. pii: 168. [Epub ahead of print]22(5):

Multi-reflecting time-of-flight mass spectrometry enables high-accuracy metabolite identification in a demonstration study using serum samples from COVID-19 patients.

Adam King, Eleanor Matthews, Garth Maker, Robert Trengove, Shaufa Shareef, Stephen Fowler, Timothy Felton, Angela Simpson, Martin Palmer, Lee A Gethings, Robert S Plumb, E N Clare Mills.

   INTRODUCTION: Accurate metabolite identification remains a major challenge in untargeted metabolomics due to overlapping signals, isomeric compounds, and insufficient mass resolution. The multi-reflecting time-of-flight (MRT) platform provides mass resolution exceeding 200,000 Full Width at Half Maximum and sub-ppm mass accuracy, enabling detailed isotopic fine structure analysis.
OBJECTIVES: The performance of the MRT platforms in identifying lipids and metabolites was evaluated using selected serum samples from a group of hospitalized COVID-19 patients.
METHODS: Lipids and polar metabolites were extracted from human serum samples from the Manchester Asthma, Allergy and Thoracic Surgery Biobank and analysed using the MRT platform with data-independent acquisition.
RESULTS: The MRT platform facilitated the resolution of overlapping lipid species and improved annotation confidence. In a pilot cohort (n = 8), metabolomic differences associated with disease severity were observed, alongside annotation of clinically relevant metabolites including sulphated bile acids and retained iohexol associated with renal dysfunction.
CONCLUSIONS: These findings demonstrate that MRT platform enhances confidence in metabolite identification and supports high-throughput metabolomics, offering significant potential for biomarker discovery in clinical studies.

Keywords:  Biomarkers; COVID-19; Lipidomics; Mass spectrometry; Metabolomics; Time-of-flight

DOI:  https://doi.org/10.1007/s11306-026-02539-w
Metabolomics. 2026 Oct 03. pii: 165. [Epub ahead of print]22(5):

Metabonaut: an open educational resource for learning reproducible, script-based untargeted LC-MS/MS metabolomics.

Philippine Louail, Marilyn De Graeve, Alexandru Mahmoud, Daniel Marques de Sá E Silva, Kozo Nishida, Vilhelm Suksi, Anna Tagliaferri, Gabriele Tomè, Vinicius Verri Hernandes, Johannes Rainer.

   BACKGROUND: Two decades after the Metabolomics Standards Initiative, public deposition of raw LC-MS/MS data into archives such as MetaboLights, Metabolomics Workbench, GNPS and MassBank has matured, but the analyses that turn raw files into reported feature tables remain difficult to share, audit and reuse. The FAIR principles, originally formulated for data, later extended to research software and to workflows, make a strong case that this gap matters; we adopt that view and ask how an educational resource can help researchers cross it.
AIM OF REVIEW: This is an educational review where we describe the principles of FAIR and reproducible analysis as they apply to untargeted LC-MS metabolomics, and we show how those principles are realised in Metabonaut, an open educational resource built on top of the R for Mass Spectrometry/Bioconductor ecosystem. Metabonaut provides executable example workflows from raw mzML files to annotated feature tables, demonstrates interoperability across programming languages, and is built with and for the community.
KEY SCIENTIFIC CONCEPTS OF REVIEW: After a brief tour of the underlying software ecosystem and of literate programming, the review discusses three topics, each through one or more Metabonaut vignettes: visual inspection and quality control, reuse of public archives at scale, and cross-language interoperability. Together, they teach how to carry FAIR practice from data to analysis: making the analysis itself, and not only the data it consumes, shareable, auditable and reusable. The source code of Metabonaut and links to pre-rendered workflow documents are available at https://github.com/RforMassSpectrometry/Metabonaut .

Keywords:   Metabonaut ; FAIR principles; Literate programming; Open educational resource; Reproducible research; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s11306-026-02538-x
Drug Des Devel Ther. 2026 ;20 632769

Simultaneous Quantification of Vedolizumab, Ustekinumab, and Infliximab in Human Serum by LC-MS/MS: Application to Therapeutic Drug Monitoring in Inflammatory Bowel Disease.

Yanbao Zhang, Na Wang, Weiyang Zheng, Ruichen Liu, Limei Li, Xianglong Zhao, Xin Zheng, Xiaohong Han.

   Purpose: With the expanding use of therapeutic monoclonal antibodies (mAbs) in inflammatory bowel disease (IBD), there is a critical need for robust, multiplex analytical tools to support therapeutic drug monitoring (TDM) and pharmacokinetic studies. This study aimed to develop and validate a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of infliximab (IFX), vedolizumab (VDZ), and ustekinumab (UST) in human serum.
Patients and Methods: A universal immunocapture strategy utilizing Protein G magnetic beads was used to enrich total IgG from human serum. Samples were subjected to tryptic digestion and analyzed in multiple reaction monitoring (MRM) mode, with rituximab as a full-process internal standard. Method validation was performed in accordance with regulatory guidelines, evaluating linearity, precision, accuracy, selectivity, recovery, matrix effect, and stability. The validated method was subsequently applied to residual serum samples from IBD patients undergoing treatment.
Results: The method demonstrated excellent linearity (R2>0.99) over the concentration ranges of 0.5-100 μg/mL for IFX and UST, and 1-200 μg/mL for VDZ. Total precision ranged from 1.1% to 14.9%, and accuracy ranged from -12.1% to 14.5%. Validation parameters, including selectivity, carryover, recovery, matrix effects, dilution integrity, and stability, were all within acceptable criteria. Analysis of the 37 clinical samples revealed marked inter-individual variability in drug concentrations. However, the absence of key covariates (eg, body weight and dosing history) precluded a formal pharmacokinetic analysis or attribution of this variability to specific clinical factors.
Conclusion: The developed LC-MS/MS method provides a practical, specific, and reproducible tool for the multiplex quantification of therapeutic mAbs. Based on a preliminary exploratory study of 37 patient samples, this method demonstrates analytical feasibility and serves as a promising tool for pharmacokinetic research; however, its definitive clinical applicability and readiness for routine TDM require further confirmation through cross-platform comparison, incurred sample reanalysis, and external validation in larger cohorts.

Keywords:  LC-MS/MS; inflammatory bowel disease; monoclonal antibodies; protein G immunocapture; therapeutic drug monitoring

DOI:  https://doi.org/10.2147/DDDT.S632769
J Pharm Biomed Anal. 2026 Oct 02. pii: S0731-7085(26)00420-6. [Epub ahead of print]283 117752

LC-MS/MS method for quantification of dupilumab in human serum using Protein G enrichment.

Kun Yang, Jianhua Wu, Chengming Yang, Liang Liu.

  Dupilumab, a monoclonal antibody targeting IL-4Rα, is used for the treatment of type 2 inflammatory diseases. Recent studies have revealed a nonlinear relationship between its serum concentration and clinical response, particularly a "biological tipping point" during dose interval prolongation, highlighting the need for individualized therapeutic drug monitoring (TDM). In this study, a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for dupilumab quantification in human serum was developed and validated. Specifically, dupilumab was enriched from 50 µL serum using Protein G magnetic beads, followed by denaturation, reduction, alkylation, and tryptic digestion. A surrogate peptide SGQSPQLLIYLGSNR was selected for quantification, and its stable isotope-labeled (SIL) analog served as an internal standard. Separation was performed on an Acquity UPLC BEH C8 column (50 mm × 2.1 mm, 1.7 µm) with detection using positive electrospray ionization in multiple reaction monitoring (MRM) mode. Excellent linearity was achieved from 1.00 to 400 µg/mL (r² > 0.99), with a lower limit of quantification of 1.00 µg/mL. Intra- and inter-run precision (%CV) and accuracy (%Bias) were within 9.27%. The validated method was successfully applied to analyze clinical serum samples. The established LC-MS/MS method provides a reliable alternative to immunoassays, with a wide dynamic range suitable for TDM and personalized dose interval adjustments in patients receiving dupilumab.

Keywords:  Dupilumab; LC-MS/MS; Monoclonal antibody; Protein G; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jpba.2026.117752
J Vis Exp. 2026 Oct 06.

A Comprehensive Dual Platform, End-to-end Workflow for Metabolic Profiling of Dried Blood Microsamples.

Daniel Marques de Sá E Silva, Marlene Thaitumu, Georgios Theodoridis, Helen Gika.

Blood microsampling (BµS) has emerged as a robust, minimally invasive alternative to traditional venipuncture, enabling the collection of low-volume specimens outside conventional clinical settings. By supporting decentralized sampling and improving patient adherence, these devices can be integrated with advanced omics technologies to facilitate large-scale and longitudinal study designs. However, successful implementation requires optimization of multiple critical steps, from sample collection and storage to extraction and analytical preparation. In this work, we propose a comprehensive end-to-end untargeted metabolomics workflow for BµS, with an emphasis on key parameters governing sample preparation and analytical performance. Volumetric absorptive microsampling (VAMS) devices were evaluated using two complementary platforms: reversed-phase liquid chromatography coupled to mass spectrometry and gas chromatography coupled to mass spectrometry. Application of the optimized workflow demonstrated broad metabolomic coverage across diverse chemical classes, highlighting the potential of VAMS-based sampling to support scalable, decentralized metabolomics studies while maintaining analytical robustness, reproducibility, and biological relevance.

DOI: https://doi.org/10.3791/72533
Anal Chem. 2026 Oct 02.

From Single Injection to Dual Insights: Integrating EAD and CID Acquisition for Spectral Library Generation and EAD-CID Spectral Matching.

Lisa Panzenboeck, Christina Brenner, Veronika Fitz, Mingxun Wang, Patrick Mueller, Evelyn Rampler, Yasin El Abiead, Gunda Koellensperger.

Mass spectral MS/MS libraries are essential in nontargeted metabolomics as they facilitate metabolite annotation and provide the basis for in silico annotation tools and fragmentation rules. While significant progress has been made toward more comprehensive MS/MS libraries, those efforts have focused mainly on collision-induced dissociation (CID)- and higher-energy collisional dissociation (HCD)-based approaches. In this work, we introduce a flow injection-high-resolution targeted MS/MS-based acquisition strategy that acquires electron-activated dissociation (EAD) and CID across a wide range of fragmentation energies within a single injection. The workflow supports both nontargeted and targeted analysis, through efficient library generation and MS/MS parameter optimization, respectively. We apply the workflow via a set of 45 steroid standards, known for their important role in human health, disease, and doping, expanding the coverage of public libraries for both CID and EAD. Finally, we utilize public MS/MS libraries and raw data to reveal that EAD MS/MS spectra can, to some extent, be annotated via existing MS/MS libraries, governed by CID and HCD, and show that the acquired CID library enables novel annotations in publicly available clinical data, demonstrating its utility for biological studies.

DOI: https://doi.org/10.1021/acs.analchem.6c01204
J Pharm Biomed Anal. 2026 Sep 16. pii: S0731-7085(26)00397-3. [Epub ahead of print]283 117729

Targeted quantification of 25 tryptophan metabolites across three metabolic pathways in serum by ultra-high-performance liquid chromatography coupled with quadrupole orbitrap high-resolution mass spectrometry.

Min Guo, Zhihua Zhang, Liping Cao, Shunhe Wang, Yue Xiao, Linlin Wang, Jianxin Zhao.

  Tryptophan metabolism encompasses three interconnected pathways, namely the kynurenine, serotonin, and gut microbiota-mediated indole pathways, which contribute to immune regulation, neural signalling, and host-microbiota interactions. Quantitative analysis of metabolites spanning these pathways remains challenging because of their diverse physicochemical properties, broad concentration ranges, and matrix interference in biological samples. In this study, a targeted analytical method based on ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) was developed for the quantification of 25 representative tryptophan metabolites in serum. Chromatographic and mass spectrometric conditions were systematically optimized, and the target metabolites were analysed using parallel reaction monitoring. All analytes showed linear responses within their respective calibration ranges, with coefficients of determination (R²) of at least 0.9990. The limits of detection and quantification ranged from 0.1 to 300 ng/mL and from 0.3 to 500 ng/mL, respectively. Intra-day and inter-day precision ranged from 0.2% to 6.9% and from 0.4% to 14.0%, respectively, while accuracy ranged from 85.2% to 117.5%. Extraction recoveries ranged from 80.6% to 108.2%, and matrix-effect values ranged from 72.9% to 119.9%, indicating analyte-dependent ion suppression or enhancement. The method was applied to serum samples from humans, rats, specific-pathogen-free mice, and germ-free mice. Comparative profiling revealed exploratory differences in the relative distributions of tryptophan metabolites among the four serum sources. Overall, the method provides broad analytical coverage of the three major tryptophan metabolic pathways and a useful approach for fit-for-purpose quantification and exploratory comparison of serum tryptophan metabolites.

Keywords:  Parallel Reaction Monitoring; Serum; Simultaneous quantitative analysis; Tryptophan metabolites

DOI:  https://doi.org/10.1016/j.jpba.2026.117729
J Sep Sci. 2026 Oct;49(10): e70547

Evaluation of a Hydrophilic Interaction Liquid Chromatography Design Space for Amino Acids.

Laura K Muehlbauer, Nahyr A López Dauphin, Timothy T Kramer, Alana Kurz, Donald S Risley.

  A column ranking system was developed for the separation of all 20 proteinogenic amino acids using hydrophilic interaction liquid chromatography mode across 18 polar stationary phases with acetonitrile/aqueous mobile phases. Each amino acid was individually evaluated on all columns using gradient elution, and the retention time data were compiled into a statistical ranking algorithm that enables selection of any analyte subset and identifies the column providing the greatest separation between successively eluting analytes. Since most of these analytes lack strong chromophores, evaporative light scattering detection was employed for quantitative detection. Mobile phase pH (3.5 to 6.0) and ammonium formate concentration (10 to 100 mM) were then optimized on the three highest-ranked columns (Accucore-150-Amide HILIC, TSKgel Amide-80, and Poroshell 120 HILIC-OH5). Retention of most amino acids was insensitive to pH, whereas arginine and lysine showed inverse dependence on buffer concentration compared with all other analytes, and 50 mM ammonium formate without pH adjustment gave the best compromise for the complete analyte set. Re-querying the ranking algorithm for smaller analyte subsets returned a different optimal column in each case, confirming that no single stationary phase is universally preferable. Knowledge gained from evaluating the design space facilitated the rapid development of a quantitative method for the determination of the amino acid composition of a capsule formulation. The isocratic assay was linear for leucine, isoleucine, and valine over approximately 0.1 to 1.0 mg/mL (R2 ≥ 0.997), recovered 102%-104% of label claim across six independent preparations, and gave injection precision of ≤ 0.45% relative standard deviation with quantitation limits of ≤ 114 µg/mL.

Keywords:  amino acids; column ranking ; column selectivity; evaporative light scattering detection; hydrophilic interaction liquid chromatography

DOI:  https://doi.org/10.1002/jssc.70547
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Sep 30. pii: S1570-0232(26)00409-5. [Epub ahead of print]1284 125320

Metabolite-targeted LC-HRMS quantification of emerging indazole-based synthetic cannabinoids in urine samples from an addiction treatment cohort.

Hacer Eroglu Icli, Zeynep Arslan, Tuna Guzide Yener Orum, Cenk Bulut, Fatih Sultan Kazdal, Seyhan Civek, Gokhan Umut, Abdullah Elci, Zeynep Turkmen.

  The continuous emergence of structurally diverse indazole-based synthetic cannabinoids (SCs) challenges routine analytical detection because parent compounds are rapidly metabolized and often absent in urine. Therefore, sensitive metabolite-targeted analytical methods are required for reliable exposure assessment. In this study, we adapted and fully validated a targeted high-resolution LC-HRMS method for the quantitative determination of eight contemporary indazole-based SC markers in human urine and applied it to 50 authentic urine specimens from treatment-seeking individuals attending an addiction treatment clinic. The method allowed the identification of robust analytical performance, with LOD and LOQ values of 0.01-0.46 ng/mL and 0.04-1.53 ng/mL, respectively, along with acceptable recovery (81.54-111.72%), bias (-4.47-13.17%), precision (RSD 5.12-15.03%), and matrix effects (75.92-100.03%). All target analytes were stable for one week at room temperature, 4 °C, and -20 °C, and no significant carryover was observed. In this treatment-seeking cohort, 76% of samples were positive for at least one SC marker, with eight markers identified across MDMB-PINACA, MDMB-BINACA, and ADB-BUTINACA analogs. MDMB-4en-PINACA butanoic acid was the dominant biomarker (62%), followed by MDMB-INACA 3,3-dimethylbutanoic acid (42%), for which this study provides the first validated quantitative urine data. Other markers were detected at lower frequencies, and frequent co-detection indicates widespread polydrug SC use. The validated high-resolution LC-HRMS method enables reliable metabolite-based assessment of exposure to emerging SCs in authentic urine samples. The predominance of MDMB-type metabolites demonstrates the applicability of the method for monitoring current trends in SC exposure, while the quantitative characterization of MDMB-INACA 3,3-dimethylbutanoic acid expands the available analytical data for newly emerging SCs. This analytical platform provides a robust tool for forensic and clinical toxicology laboratories to monitor SC exposure in rapidly evolving drug markets.

Keywords:  ADB-BUTINACA; MDMB-4en-PINACA; MDMB-BINACA; Metabolite profiling; Novel psychoactive substances; Synthetic cannabinoids

DOI:  https://doi.org/10.1016/j.jchromb.2026.125320
Nanoscale. 2026 Oct 06.

Nanomatrix engineering for LDI-MS based biomedical metabolomics.

Yi Gu, Yanxi Yang, Jiao Wu, Kun Qian.

Metabolomics provides functional molecular readouts of disease phenotypes, therapeutic responses, and tissue microenvironments, but its extension to low-volume, single-cell, and spatially resolved samples remains analytically challenging. Laser desorption/ionization mass spectrometry (LDI-MS) offers a rapid, direct, and high-throughput route for small-molecule analysis. However, its performance in biomedical metabolomics is largely governed by the matrix-analyte interface. Nanomaterials have emerged as attractive LDI-MS matrices because of their low background in the low-mass region, large surface area, tunable photothermal and electronic properties, and designable surface chemistry. In this review, we summarize recent advances in nanomatrix-assisted LDI-MS for biomedical metabolomics, emphasizing design principles. We discuss three key design dimensions of nanomatrix and interface engineering: structural engineering for energy absorption and desorption efficiency, surface functionalization for metabolite enrichment and low-interference ionization, and interface engineering for reproducibility, throughput, and low-volume sample compatibility. Representative biomedical applications are highlighted, including biofluid metabolic fingerprinting, extracellular vesicle and single-cell metabolomics, and spatial metabolic imaging. Finally, we discuss current challenges in matrix reproducibility, metabolite coverage, workflow standardization, and clinical validation, and outline future opportunities for integrated nanomatrix platforms in multiscale biomedical metabolomics.

DOI: https://doi.org/10.1039/d6nr02874h
Anal Bioanal Chem. 2026 Oct 08.

Orthogonality-driven offline two-dimensional liquid chromatography coupled with high-resolution mass spectrometry for comprehensive chemical profiling and quality marker screening of Renshen Guben oral liquid.

Dong Wang, Mengrong Li, Xiangyu Zhang, Tianshu Liu, Liming Wang, Bingjie Zhu, Guimin Zhang.

  Traditional Chinese patent medicines present formidable analytical challenges due to their multi-herb composition, extreme polarity diversity, and vast concentration disparities among constituents, rendering conventional one-dimensional liquid chromatography-mass spectrometry (1D LC-MS) insufficient for comprehensive quality assessment. This study establishes an orthogonality-driven offline two-dimensional liquid chromatography system (HILIC × RPLC) coupled with Q-Orbitrap mass spectrometry for in-depth chemical profiling and rational quality marker (Q-marker) screening of Renshen Guben oral liquid (RSGB), a clinically used immunomodulatory formulation comprising ten herbal ingredients. The 2D-LC platform was systematically optimized and validated, achieving a ~50-fold improvement over 1D LC-MS in component coverage. Global natural products social molecular networking (GNPS) was employed to visualize MS/MS spectral similarity relationships, facilitating confident annotation of structurally related analogues across 13 chemical classes. A total of 260 compounds were identified, among which 48 were verified against reference standards. Building upon this comprehensive chemical foundation, a multidimensional Q-marker screening strategy was devised by integrating network pharmacology prediction, molecular docking evaluation against the core immune-related target HSP90AA1, and chromatographic fingerprint correlation of ten constituent herbs, ultimately designating 12 compounds from eight distinct herbs as Q-markers. To address the wide dynamic range in component abundance, an adjusted multiple reaction monitoring (MRM) method utilizing triple quadrupole mass spectrometry was developed, enabling simultaneous quantification spanning three orders of magnitude. Method validation demonstrated excellent linearity (R2 > 0.999) and satisfactory precision and repeatability for all 12 Q-markers. Quantitative analysis of ten commercial batches revealed significant inter-batch variations, with gallic acid as the most abundant constituent and alisol B the least, underscoring the necessity of multicomponent quality control. This integrated workflow bridges the gap between comprehensive chemical characterization and pragmatic quantitative assessment, offering a transferable paradigm for holistic quality evaluation of complex botanical formulations.

Keywords:  High-resolution mass spectrometry; Molecular networking; Quality marker; Renshen Guben; Two-dimensional liquid chromatography

DOI:  https://doi.org/10.1007/s00216-026-06857-9
J Sep Sci. 2026 Oct;49(10): e70539

Recent Advances in Sample Preparation for Cholesterol and Oxysterol Analysis in Complex Biological Matrices.

Haizhen Zhang, Longchan Liu, Haoyue Zhang, Linnan Li, Zhengtao Wang, Li Yang.

  Cholesterol and oxysterols are key molecules that regulate physiological processes and participate in the occurrence and progression of various major diseases. Accurate and efficient analytical detection of these substances in complex biological matrices is critical for disease mechanism research and biomarker discovery. However, challenges including complex biological matrices, large differences in analyte abundance, susceptibility to auto‑oxidation, low ionization efficiency, and difficulty in isomer separation pose severe obstacles to reliable analysis, making sample preparation the core step for accurate quantification and valid identification. Continuous innovations have been achieved in sample preparation techniques, such as liquid-liquid extraction, solid‑phase extraction, deep eutectic solvent extraction, molecularly imprinted extraction, derivatization, as well as integrated and automated workflows. These advances improve extraction efficiency, selectivity, antioxidant protection, and throughput, while effectively reducing matrix interference and artificial oxidation. This paper systematically reviews the research progress in sample preparation for cholesterol and oxysterol analysis in complex biological matrices from 2015 to 2026, highlights the technical advantages, application scenarios, and current limitations of various pretreatment strategies, and prospects the development trends toward greening, intelligence, and high throughput.

Keywords:  biological matrices; cholesterol; extraction; oxysterols; sample preparation

DOI:  https://doi.org/10.1002/jssc.70539
Anal Methods. 2026 Oct 09.

Lipid profiling of meat samples using surface extraction followed by multiple reaction monitoring profiling.

Gislaine Dos Santos, Tiago J P Sobreira, Sage Y Min, Bruce R Cooper, Ryan T Hilger, Christina R Ferreira.

Direct sample extraction methods suitable for lipids such as desorption electrospray ionization (DESI), rapid evaporative ionization mass spectrometry (REIMS), and liquid extraction surface analysis (LESA) are combined with ionization or direct sample delivery to a mass spectrometer. Here, we describe three types of off-line direct sample extraction methods and their combination with multiple reaction monitoring (MRM) profiling to survey lipids present on the surface of beef and chicken meat as proof-of-concept samples. A collection of 679 MRMs selected from over 3200 MRMs related to lipids was profiled using a conventional capillary binary pump, autosampler and a triple quadrupole mass spectrometer system. The surface extraction experiments were performed manually by simply dispensing solvent onto the surface of the samples and recovering it immediately (liquid surface extraction - LSE), or by using solvent loaded into a large orifice tip to contact the sample for 5 seconds without dispensing (solvent "touch" extraction - STE). The third approach, surface vaporization extraction (SVE), was based on extracting vapors from the surface of the sample created using the tip of a wood-burning tool, collecting the vapors in a microtube, and diluting them for flow injection. LSE was used to evaluate the effect of different solvent compositions on the number of MRMs detected. Solvent acidification was also evaluated. Compared to the Bligh & Dyer extraction method using homogenized beef samples, LSE provided a consistently lower number of MRMs detected and the most informative solvent for surface extraction was pure acetonitrile. Since meat samples' surfaces are not completely flat and also present natural irregularities, the solvent volume recovered was variable. Therefore, we turned to STE to evaluate whether the lipid composition of beef brown spots was different from the red regions and to probe changes in composition due to one freeze-thaw cycle. Also, the reproducibility of STE between three users was evaluated. The third surface extraction approach, SVE, was applied to study the lipid profiles of vapors collected from raw and cooked meat. Overall, the three surface methods were able to provide informative discrimination for the different aspects assayed.

DOI: https://doi.org/10.1039/d6ay01407k
bioRxiv. 2026 Aug 13. pii: 2026.08.12.744242. [Epub ahead of print]

Integrated assessment of fatty acid metabolism and cellular energy status using HILIC-MS/MS.

Mariana Lopes, Kyle D Roberts, Alexis E Heath, Peder J Lund.

Acetyl-CoA and other acyl-CoA thioesters are critical intermediates in the metabolic reactions that cells rely on to produce energy and carry out biosynthesis. Therefore, the analysis of acyl-CoA provides valuable information about the metabolic activity of cells, especially when combined with stable isotope tracing. Acyl-CoA species are routinely monitored by reversed-phase liquid chromatography coupled to tandem mass spectrometry (RPLC-MS/MS). However, drastic differences in the hydrophobicity of short-chain versus long-chain acyl-CoA species have been challenging to accommodate with a single set of RPLC conditions. Here, we describe a convenient method based on hydrophilic interaction liquid chromatography (HILIC-MS/MS) for the concurrent detection of both short-chain and long-chain acyl-CoA and their corresponding acyl-carnitine species. Using this strategy, we tracked the metabolism of isotope-labeled fatty acids in multiple cell lines, which revealed differences in their propensities for fatty acid oxidation and the extent to which isotope incorporation into acyl-CoA mirrored that of acyl-carnitine. We also applied the HILIC-MS/MS workflow to the analysis of NADH and ATP, making it a useful technique for gauging cellular bioenergetics as reflected by the acetyl-CoA/CoA, NADH/NAD + , and ATP/ADP ratios. Altogether, this HILIC-MS/MS platform enables a streamlined analysis of acyl-CoA species and other key intermediates in cell metabolism.

DOI: https://doi.org/10.64898/2026.08.12.744242
Food Chem. 2026 Oct 05. pii: S0308-8146(26)03625-3. [Epub ahead of print]532(Pt 1): 151465

Phenolic-focused untargeted ion mobility LC-MS metabolomics reveals season-associated chemical signatures in Scottish honey.

Renata Garbellini Duft, Julian L Griffin.

  Honey authenticity is compromised by mislabelled origin, blending, and product modification, undermining traceability and consumer trust. To address this, we developed a phenolic-focused, untargeted ultra-high-performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC-IM-QTOF-MS) workflow for profiling Scottish honeys using a low-solvent extraction approach and collision cross section-supported annotation (CCS-SA). Thirty-nine samples, including Scottish heather, spring, and summer honeys together with market-relevant comparators, were analysed to investigate compositional variation linked to origin, season, and product type. The workflow produced a robust, quality-controlled feature set with strong dilution linearity, while integration of accurate mass, retention time, MS/MS, and CCS increased confidence in marker annotation. Paired within-apiary comparison provided robust evidence that spring and summer honeys carry distinct season-associated chemical signatures, and heather honey emerged as a chemically distinct class. Exploratory analyses also indicated differences between Scottish and non-Scottish honeys and compositional shifts associated with blending and infusion. Because several comparator groups were small and unbalanced, these findings are considered preliminary. Overall, the workflow provides a robust platform for characterising seasonal variation and discovering candidate authenticity markers in Scottish honey.

Keywords:  Collision cross-section; Food fraud; Honey authenticity; Ion mobility; Metabolomics; Phenolic compounds; Scottish honey

DOI:  https://doi.org/10.1016/j.foodchem.2026.151465
J Dairy Sci. 2026 Oct 09. pii: S0022-0302(26)03347-3. [Epub ahead of print]

Comprehensive evaluation of metabolite imputation methods and the effect of imputation accuracy on the performance of metabolite-trait association using large-scale metabolite datasets in cattle.

Zhujun Chen, Jun Teng, Chongwei Duan, Xinyi Zhang, Jian Yang, Fen Pei, Xiuxin Zhao, Guanghui Xue, Yan Liu, Huiming Liu, Hongding Gao, Rongling Li, Yundong Gao, Jianbin Li, Qin Zhang, Xiao Wang.

  As metabolomics data are typically acquired across multiple batches over months or even years, the integrated metabolite data sets often face complex missing-value challenges arising from fluctuations in instrument performance, variation in detection windows, and heterogeneity among analytical platforms. Accurate imputation of missing values is therefore a crucial strategy to addressing these challenges and enhancing the performance of downstream metabolomics analyses. In this study, we utilized large-scale authentic plasma and serum metabolite data sets (n = ∼3,000 metabolites) in Holstein cattle (n = ∼3,000 individuals), with each data set consisting of 2 batches, to evaluate the performance of 5 imputation methods (MIRTH, PIXANT, EM, KNN, and RF) to impute missing metabolites within batches and non-overlapping metabolites between batches. We demonstrated that RF yielded the best imputation performance in most cases, with accuracies reaching approximately 0.6 ∼0.7 for within-batch imputation and approximately 0.3 ∼0.4 for cross-batch imputation. Subsequently, we assessed the effect of imputation accuracy on the performance of metabolite-trait association analysis for 5 milk production traits using RF-imputed metabolite data. Compared with significantly associated metabolites identified using the original metabolite data, analysis using the imputed metabolite data resulted in substantial reductions in statistical power as well as increases in false-positive associations, particularly when imputed data from different batches were merged. Further efforts are needed to improve the accuracy for cross-batch imputation by developing new imputation methods and increasing the number of overlapping metabolites across batches.

Keywords:  cattle; imputation methods; metabolite-trait association; plasma and serum metabolite; within-batch and cross-batch datasets

DOI:  https://doi.org/10.3168/jds.2026-28962
Chem Sci. 2026 Sep 04.

Universal charge-tag labelling of undetectable polymers for mass spectrometric characterization.

Charles Killeen, Liliia Pestereva, Maxine A Turner, Luca Swainson, Jeremy E Wulff, J Scott McIndoe.

Despite massive advances in mass spectrometric technologies, there remains a class of materials largely impervious to analysis: high molecular weight aliphatic hydrocarbons. Electrospray ionization mass spectrometry (ESI-MS) is used extensively as a tool in petroleomics to identify and fingerprint the polar constituents of oil, but the non-polar constituents remain challenging to analyze with ESI-MS due to poor ionization efficiency. Derivatization methods which bond electrospray-active tags to analytes impart charge and increase analyte response and have been developed for many different classes of petrochemicals, but not hydrocarbons. In this work, a method is described which renders aliphatic hydrocarbons amenable to ESI-MS analysis through C-H insertion into a substrate molecule of a diazirine-derived carbene, which is bound to a pendant alkyne. Subsequent electrospray-tagging occurs through copper-catalyzed azide alkyne cycloaddition, appending a triphenylphosphonium cation to the derivatized compound. The process was developed and benchmarked with cyclododecane as a model substrate to evaluate tagging efficiency and product distribution and then applied to characterize several commercial products composed of aliphatic compounds. While derivatization efficiency and ionization response may vary across substrates, the method provides a complementary route to established ionization techniques by enabling hydrocarbon analysis within ESI-based high-resolution mass spectrometric workflows. Its primary utility lies in enabling ESI-compatible fingerprinting of unfunctionalized compounds.

DOI: https://doi.org/10.1039/d6sc05830b
J Sep Sci. 2026 Oct;49(10): e70549

Green Sorptive Sampling Coupled With UHPLC-IMS-MS and GC×GC-TOFMS for Characterization of Migrating Substances From Plastic Food Contact Materials.

Gizelle Roque van Niekerk, Yvette Naudé, Madelien Wooding.

  Comprehensive analysis of migrating compounds from plastic food contact materials (FCMs) is analytically demanding due to the structural diversity and broad physicochemical properties of intentionally and non-intentionally added substances (IAS and NIAS). This necessitates the development of advanced, multidimensional separation techniques coupled with high-resolution detection for trace-level characterization. Five million tones of chemical additives are incorporated into plastic FCMs annually, which is concerning considering the health risks associated with human exposure to migrating chemicals. This study provides a simplified green sorptive sampling loop for detecting and quantifying IAS and NIAS that migrate from FCMs during everyday use. A polydimethylsiloxane (PDMS) sorptive sampler loop was used as the sampling device, allowing migrated compounds to be enriched directly from the sample matrix. After extraction, the same sampler format was compatible with two analytical workflows: thermal desorption directly into the GC inlet for GC×GC-TOFMS analysis, or liquid back-extraction into solvent for subsequent UHPLC-IMS-MS analysis. Complementary chromatography and ion mobility high-resolution mass spectrometry (UHPLC-IMS-MS and GC×GC-TOFMS) enabled the detection and quantification of a range of chemical compounds at trace levels, including phthalates, UV absorbers, stabilizers, surfactants, and hydrocarbons. Over 30 compounds were detected as migrating from FCMs using GC-MS, and 26 compounds were identified using LC-MS. Unknown target identification using LC-MS was aided by the use of collision cross section (CCS) values, thus strengthening putative identification. Limits of detection of the eight target analytes ranged from 0.023 to 97 ng L-1 for LC-MS and IAS were detected at concentrations ranging from 0.0065 to ∼25 µg L- 1. Our study exemplifies the wide range of chemical compounds that migrate from FCMs during everyday FCM use. The green sampling technique presented here allowed the quantification of daily exposure to these chemicals and has potential application to human health and environmental risk assessments.

Keywords:  collision cross section; extractables and leachables; food contact materials; plasticizers; sorptive extraction

DOI:  https://doi.org/10.1002/jssc.70549
Clin Chem Lab Med. 2026 Oct 09.

Analytical performance and clinical classification value of a novel sandwich immunoassay for total testosterone measurement in low-concentration and hemodialysis populations.

He Shi, Huimei Wei, Shuaiwei Han, Minjie Liu, Bingjie Shui, Jijuan Gao, Yu Luo, Huaying Zhang, Huiai Lu, Pengcheng Tong, Huan He, Jianguo Wu.

   OBJECTIVES: To evaluate the analytical performance and clinical classification value of a novel sandwich testosterone immunoassay, particularly for biochemical testosterone elevation in women and deficiency in male hemodialysis patients, using LC-MS/MS as the reference method.
METHODS: A total of 387 residual clinical serum samples were compared using Mindray TESTO, Siemens TSTII, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among these, 226 had testosterone concentrations <1.0 ng/mL, and 133 reproductive-age women were evaluated for biochemical hyperandrogenism classification. An additional 119 hemodialysis samples (94 men, 25 women) were assessed, with the 94 men evaluated for biochemical testosterone deficiency classification. Analyses included Passing-Bablok regression, Bland-Altman analysis, and independent imprecision, linearity, and interference studies.
RESULTS: The Mindray TESTO assay showed total CVs of 1.68-2.71 % and good linearity (r=0.9997). Cross-reactivity was generally low, although phenylbutazone produced biases exceeding ±10 %. Both immunoassays correlated strongly with LC-MS/MS, with Mindray TESTO showing higher correlation and narrower limits of agreement in low-concentration samples (r=0.9603 vs. 0.8920). In women of reproductive age, Mindray TESTO demonstrated higher sensitivity (88.89 vs. 11.11 %) and κ agreement (0.784 vs. 0.189) for biochemical hyperandrogenism classification. In male hemodialysis patients, it achieved higher accuracy (94.68 vs. 88.30 %) and κ agreement (0.838 vs. 0.680) for biochemical testosterone deficiency classification. The ROC AUCs did not differ significantly (0.966 vs. 0.956; p=0.2776).
CONCLUSIONS: The novel sandwich immunoassay showed improved agreement with LC-MS/MS at low testosterone concentrations and favorable performance in classifying biochemical testosterone elevation and deficiency.

Keywords:  analytical performance; chemiluminescence immunoassay; hemodialysis; liquid chromatography–tandem mass spectrometry; testosterone

DOI:  https://doi.org/10.1515/cclm-2026-0886