bims-metlip 2026-09-27 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–09–27
thirty-two papers selected by
Sofia Costa, Matterworks

Development and Validation of an LC-MS/MS-Based Screening Method for Determination of Salbutamol in Urine for Doping Control Using a Surrogate Analyte Approach with Stable Isotope-Labeled Salbutamol (Salbutamol-d9).
SIL-IS LC-MS/MS method for rapid quantification of phloroglucinol in human plasma: development, validation and application to a pharmacokinetic study.
MStargetR: a reproducible, containerised workflow for end-to-end targeted (MRM/SRM) mass spectrometry data processing in R.
Design of experiments-driven optimization of dual ultra-high-performance liquid chromatography high resolution mass spectrometry for integrated fecal metabolomics and lipidomics.
Synthesis-driven reverse metabolomics reveals 3-hydroxy N-acyl amides as gut microbial molecules.
Simultaneous hydrophilic interaction liquid chromatography-tandem mass spectrometry quantification of serum neurotransmitters and related metabolites: Development, validation, and application to antenatal depression.
A high-throughput LC-MS/MS assay for therapeutic drug monitoring of combined immunosuppressive and antimicrobial therapy.
Development, Validation, and Clinical Application of a Simultaneous Quantification Method of Seventeen Antituberculosis Drugs in Plasma, Serum, or Saliva Using LC-MS/MS.
Development and Validation of an LC-MS/MS Method for the Detection of Substituted Phenethylamines and Phenidates Designated as Temporary Narcotics in Urine.
Preparation of Deuterium-Labeled Tadalafil and Omeprazole by Carbon Hydrogen-Deuterium Exchange: Hydrogen Scrambling, Acid-Induced Transformation, and LC-MS Applications.
Position specific 15N isotope analysis at natural abundance by 1H NMR.
Optimization of untargeted lipidomics analysis of human plasma after Dangguijakyak-san administration.
Determination of 25 Organophosphate Ester Flame Retardants in Soils by Accelerated Solvent Extraction-Ultra-High Performance Liquid Chromatography.
Multiclass Cyanotoxins in Aquatic Environments: Occurrence, Impacts and Detection Techniques.
A complementary dual-platform analytical strategy for resolving matrix effects and stability limitations in bioanalysis.
Plant-based dietary biomarkers: from discovery to validation using mass spectrometry-based metabolomics.
Quantification of Amlexanox in Beagle Dog Plasma by LC-MS/MS and Its Application in a Comparative Pharmacokinetic Study of Conventional and Sustained-Release Tablets.
Per- and Polyfluoroalkyl Substances (PFAS) in Michigan: A Novel High-Throughput Method with Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis of 39 PFAS Compounds in Human and Bovine Dried Blood Spots (DBS).
Metabodeconplus-An R Package for Automated Deconvolution and Alignment of 1D NMR Metabolomics Data.
Systematic mass-spectrometry-guided metabolic fingerprinting elucidates diversity of specialized metabolites across the Brassicaceae.
Comprehensive Comparison of Three Automated 25-Hydroxyvitamin D Immunoassays with a VDSCP Traceable LC-MS/MS Method Across Clinically Relevant Decision Thresholds.
Organ-Resolved Untargeted LC-MS/MS Profiling of Putative Metabolite Families in Aerial Organs of Vitex negundo L.
Hyphenation of supercritical fluid chromatography to high resolution Orbitrap mass spectrometry for the purpose of non-targeted small molecule analysis.
Instant CMQT stabilization coupled UHPLC-MS/MS for the quantification of reduced and total forms of cysteine, homocysteine, and glutathione in blood plasma.
Assembling an Expanded Metabolome for Camu-Camu (Myrciaria Dubia): Bioactive and Nutritional Compounds Relevant to Functional Food Research.
Comparative 1H-NMR profiling of plasma and serum metabolites in giraffes and white rhinoceroses.
Validated LC-MS/MS Methods for Quantification of AC02 and Porcine ACTH1-39 in Human Plasma: Application to a Phase I Study.
Chemical Derivatization Strategies for Expanding Small Biomolecule Analysis by MALDI-MS.
Tissue expansion mass spectrometry imaging (TEMI) for high-spatial-resolution multiomics molecular mapping.
A Derivatization-Free GC-MS Method for Reliable Quantification of Short-Chain Fatty Acids in Sparus aurata.
Optimized Sodium-Enhanced MALDI-2 Enables Multimodal Imaging of Triacylglycerols.
Supercritical Fluid Chromatography for Expedited Sample Clean-Up in Isotope Ratio Mass Spectrometry Including Cholesterol as New Endogenous Reference Compound.

Iran J Pharm Res. 2026 Jan-Dec;25(1):25(1): e169531

Development and Validation of an LC-MS/MS-Based Screening Method for Determination of Salbutamol in Urine for Doping Control Using a Surrogate Analyte Approach with Stable Isotope-Labeled Salbutamol (Salbutamol-d9).

Zeinab Srour, Farzad Kobarfard, Zahra Sheikholislam, Mehrdad Faizi, Bahram Daraei.

   Background: Athletic doping has long received attention in the sports community. Salbutamol is prohibited by the World Anti-Doping Agency (WADA) both in and out of competition, with a urinary concentration threshold of 1000 ng/mL defining the legal limit. Traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical methods that use an internal standard cannot fully overcome matrix effects and biological variation, requiring revalidation for different biological samples; this process can be time-consuming and can remain prone to matrix-related errors.
Objectives: To develop a novel approach for screening salbutamol in urine that overcomes the limitations of traditional LC-MS/MS methods and improves analytical reliability and efficiency across biological matrices.
Methods: A surrogate analyte-based LC-MS/MS method was developed for the quantitative determination of salbutamol in human urine, using salbutamol-d9 as a stable isotope-labeled surrogate analyte. During method optimization, concentrations ranging from 1 ppm to 0.1 ppb were analyzed in aqueous and urine samples to evaluate extraction efficiency, purification, and matrix effects. Molar-equivalent concentrations of unlabeled salbutamol (1000 ng/mL, the WADA legal limit) and labeled salbutamol (1039 ng/mL) were analyzed in both matrices to validate the surrogate analyte approach and confirm comparable peak areas. To evaluate the applicability of the method, urine samples were collected from healthy volunteers after oral administration of 4 mg salbutamol. All samples were extracted under alkaline conditions using liquid-liquid extraction and analyzed by LC-MS/MS in positive-ion mode with multiple reaction monitoring (MRM). The labeled compound served as a reference for interpretation of the results. Method validation was performed according to international guidelines, including assessments of linearity, precision, accuracy, and sensitivity.
Results: The method demonstrated excellent linearity (R2 > 0.99), with intra- and inter-day precision within 8% RSD and acceptable accuracy. The limits of detection (LOD) and quantification (LOQ) were 0.3 and 1 ng/mL, respectively. Analysis of urine from healthy volunteers following a 4 mg oral dose confirmed reliable quantification for up to 24 hours, demonstrating applicability to biological matrices.
Conclusions: These findings indicate that this isotope-based surrogate analyte approach eliminates the need for calibration curves, compensates for matrix effects and sample preparation errors, and improves analytical reliability and efficiency across biological matrices.

Keywords:  Doping Control; LC-MS/MS; Labeled Analyte; Salbutamol; Surrogate Analyte

DOI:  https://doi.org/10.5812/ijpr-169531
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Sep 09. pii: S1570-0232(26)00387-9. [Epub ahead of print]1284 125298

SIL-IS LC-MS/MS method for rapid quantification of phloroglucinol in human plasma: development, validation and application to a pharmacokinetic study.

Qiqi Sun, Xianghong Li, Yujie Dong, Jie Shen, Haitang Xie, Yuanwei Jia.

  Phloroglucinol is a smooth muscle relaxant widely used for the symptomatic treatment of gastrointestinal, biliary, and genitourinary spasms. A sensitive, rapid, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method incorporating a stable isotope-labeled internal standard (SIL-IS, phloroglucinol-13C6) was developed and validated for the quantification of phloroglucinol in human plasma. Plasma samples were prepared using a simple liquid-liquid extraction (LLE) procedure in a 96-well format and analyzed on a reversed-phase phenyl column with a total run time of 4.5 min. The analyte and the stable isotope-labeled internal standard were monitored by multiple reaction monitoring (MRM) in negative electrospray ionization (ESI) mode on a triple quadrupole mass spectrometer, with transitions of m/z 125.0 → 57.0 and m/z 131.0 → 60.1, respectively. The method demonstrated excellent linearity over the concentration range of 0.750-750 ng/mL with a lower limit of quantification (LLOQ) of 0.750 ng/mL. The validation was conducted in strict accordance with the requirements of bioanalytical guidelines, encompassing assessments of selectivity, linearity, accuracy and precision, recovery, matrix effect, stability, and dilution integrity. The results demonstrated that all validation parameters met the predefined acceptance criteria. It was successfully applied to a pharmacokinetic and bioequivalence study of phloroglucinol orally disintegrating tablets in healthy Chinese volunteers under fasting and fed conditions, enabling reliable characterization of pharmacokinetic profiles.

Keywords:  Human plasma; LC–MS/MS; Method validation; Pharmacokinetics; Phloroglucinol

DOI:  https://doi.org/10.1016/j.jchromb.2026.125298
Metabolomics. 2026 Sep 25. pii: 160. [Epub ahead of print]22(5):

MStargetR: a reproducible, containerised workflow for end-to-end targeted (MRM/SRM) mass spectrometry data processing in R.

Harrison Szemray, Vimalnath Nambiar, Dana Hicks, Samantha Lodge, Julien Wist, Nathan G Lawler, Luke Whiley.

   INTRODUCTION: Targeted metabolic phenotyping by liquid chromatography-tandem mass spectrometry (LC-MS/MS) relies on a fragmented toolchain of proprietary vendor formats, manual integration steps, and ad hoc quality-control (QC) scripts, introducing user- and laboratory-level variation that undermines reproducibility and confounds cross-laboratory and retrospective comparison.
OBJECTIVES: To provide an open-source, R-native workflow for targeted multiple reaction monitoring (MRM/SRM) mass spectrometry data that consolidates vendor file conversion, peak integration, and QC reporting into a single reproducible pipeline while preserving auditable, human-in-the-loop peak review.
METHODS: MStargetR builds on msConvert and Skyline through three modules: msConvertR (vendor-to-mzML conversion), PeakForgeR (peak boundary optimisation and automated peak integration executed through Skyline), and qcCheckR (normalisation, concentration calculation, signal and batch correction, and reporting). Additionally, MStargetR has a standalone correction module and a Shiny graphical user interface. Third-party tools are pinned in version-controlled Docker images (with Apptainer support for high-performance computing), and each analytical plate emits a fully populated sky document for inspection and reimport.
RESULTS: Applied to a published targeted lipidomics dataset of 128 human plasma samples targeting 1,161 lipid species, MStargetR processed all samples end-to-end, recovering all 1,161 targeted lipid features, 949 of which (81.7%) were detected and returned RSD < 30% across replicated long-term reference QCs. Analysis scaled linearly to 4,200 samples, averaging 4.1 s per sample.
CONCLUSION: MStargetR delivers automated batch processing, auditable peak review, and a documented QC layer in a single reproducible pipeline, supporting FAIR-aligned targeted metabolomics.

Keywords:  Lipidomics; Mass spectrometry; Multiple reaction monitoring; Phenotype; Precision medicine; Reproducibility of results; Software

DOI:  https://doi.org/10.1007/s11306-026-02541-2
Anal Chim Acta. 2026 Nov 08. pii: S0003-2670(26)01037-8. [Epub ahead of print]1422 346087

Design of experiments-driven optimization of dual ultra-high-performance liquid chromatography high resolution mass spectrometry for integrated fecal metabolomics and lipidomics.

Tiffany De Troyer, Kimberly De Windt, Beata Pomian, Ellen De Paepe, Vera Plekhova, Lynn Vanhaecke.

  Recently, feces has gained increased attention in metabolomics and lipidomics research due to its ability to reflect complex diet-host-microbiome interactions. Traditionally, these analyses rely on separate workflows, resulting in longer analysis times and increased instrument load. To address these limitations, we present a dual ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (dual UHPLC-HRMS) approach. As a first step, two previously validated single UHPLC-HRMS methods for metabolomics and lipidomics, each demonstrating robust chromatographic separation of compounds, covering a broad physicochemical range (LogP -5.30 to 21.90), were selected. To integrate both workflows into the dual platform, thirteen critical LC-MS parameters were systematically optimized using a Design of Experiments (DoE). The parameters encompassed ion generation, transmission and detection, injection-related factors, unified column oven conditions, and source geometry. This approach enabled a robust dual workflow, achieving a 21% reduction in analysis time. Targeted evaluation demonstrated consistent detection of 287 metabolites (260 with CV<20%) and 162 lipids (144 with CV<20%), thereby outperforming the single methods, which detected 272 metabolites (232 with CV<20%) and 145 lipids (116 with CV<20%), respectively. Untargeted analysis further demonstrated increased coverage, with an additional 1652 metabolite and 3966 lipid features detected with the dual method without compromising repeatability of the feature signal intensities (79.4% vs. 81.2% for metabolomics and 87.4% vs. 82.7% for lipidomics, with CV<30%). Our novel dual UHPLC-HRMS workflow enhances analytical throughput, while also improving fecal metabolome and lipidome coverage and repeatability, offering a robust and cost-effective solution for large-scale studies.

Keywords:  Analytical method development; Gut phenotyping; Integrated multi-omics; Small molecules; Statistical design; Stool

DOI:  https://doi.org/10.1016/j.aca.2026.346087
mSystems. 2026 Sep 24. e0083926

Synthesis-driven reverse metabolomics reveals 3-hydroxy N-acyl amides as gut microbial molecules.

Victoria Deleray, Vincent Charron-Lamoureux, Kyle Vittali, Helena Mannochio-Russo, Crystal X Wang, Corinn Walker, Karsten Zengler, Dionicio Siegel, Pieter C Dorrestein.

  3-Hydroxy N-acyl amides are bioactive lipids with reported anti-obesity and glucose-regulating effects, yet they are rarely detected in untargeted metabolomics studies because they are largely absent from existing spectral reference libraries. To address this gap, we synthesized an MS/MS spectral resource comprising 436 structurally diverse 3-hydroxy N-acyl amides, spanning 3- to 18-carbon chains with a wide range of amine headgroups, such as ornithine, valine, and dopamine. Using a synthesis-driven reverse metabolomics approach, we found 161,626 spectral matches across 54,744 publicly available files in untargeted metabolomics data sets, revealing widespread occurrences in biological samples, including human-derived specimens. Of these molecules detected through MS/MS spectral matching, 334 represent newly reported biological entities. We further confirmed their presence in human saliva, stool, and skin using retention time and ion mobility measurements. Frequent detection in microbial data sets and validation in communities of human-derived gut bacteria support microbial production. Several metabolites also showed altered abundance in individuals with diabetes mellitus, showing that this lipid class is modulated in human metabolic disease. Together, these findings establish 3-hydroxy N-acyl amides as a distinct and biologically relevant lipid class, and the accompanying MS/MS spectral resource will enable their broader recognition and study in untargeted metabolomics data.
IMPORTANCE: Microbe-host interactions lead to the production of metabolites that play an important role in human health and disease, though the annotation of such metabolites remains scarce. We created a re-usable MS/MS spectral library of an underrepresented class of compounds-3-hydroxy N-acyl amides. Our study characterized the presence of over 400 3-hydroxy N-acyl amides among thousands of public untargeted metabolomics datasets. We found over 100,000 instances of these metabolites within microbial, plant, and animal data, and used orthogonal validation methods to confirm nine previously unreported molecules within human feces, saliva, and skin samples. We cultured communities of human-derived gut bacteria and found evidence of bacterial production of four 3-hydroxy N-acyl amides. Our results suggest that these metabolites play important roles in human metabolism and disease, and sharing this curated resource enables the detection of these metabolites in future studies.

Keywords:  diabetes; ion mobility; mass spectrometry; microbial metabolism; reverse metabolomics

DOI:  https://doi.org/10.1128/msystems.00839-26
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Sep 22. pii: S1570-0232(26)00399-5. [Epub ahead of print]1284 125310

Simultaneous hydrophilic interaction liquid chromatography-tandem mass spectrometry quantification of serum neurotransmitters and related metabolites: Development, validation, and application to antenatal depression.

Rui Guo, Miao Zhu, Fei Huang, Feng Cheng, Zhonghua Lu, Song Xu, Li He, Guang Xu, Heng Wang, Jie Zhang.

  Serum neurotransmitter profiling is analytically challenging because the target panel includes highly polar compounds with markedly different ionization behaviors, while several catecholamine-related analytes are also susceptible to oxidation during thawing and handling. Here, we developed and validated a derivatization-free hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 15 serum neurotransmitters and related metabolites within a 12-min run. To improve the quantitative reliability of oxidation-prone analytes, serum samples were stabilized immediately after thawing with ascorbic acid before protein precipitation. The final workflow combined hydrophilic interaction liquid chromatography separation, stable isotope dilution, and single-step protein precipitation. The method showed good linearity across the validated concentration ranges for all analytes, with coefficients of determination above 0.99, limits of detection of 0.01-0.30 ng/mL, recoveries of 85.19% to 107.41%, and acceptable intra- and inter-day precision. Stability experiments further demonstrated marked signal loss for labile catecholamine-related analytes in the absence of antioxidant protection, whereas immediate post-thaw ascorbic acid stabilization substantially improved preservation of dopamine, norepinephrine, epinephrine, serotonin, and L-dopa during sample handling. Application of the method to serum samples from 188 pregnant women identified higher gamma-aminobutyric acid together with lower acetylcholine, choline, norepinephrine, L-dopa, serotonin, and 3-methoxytyramine in the antenatal depression group than in non-depressed controls. These findings support the use of this stability-aware hydrophilic interaction liquid chromatography-tandem mass spectrometry platform for cohort-scale serum neurotransmitter profiling and biomarker-oriented studies in pregnancy.

Keywords:  Antenatal depression; Hydrophilic interaction liquid chromatography; Mass spectrometry; Neurotransmitters; Serum

DOI:  https://doi.org/10.1016/j.jchromb.2026.125310
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Sep 17. pii: S1570-0232(26)00392-2. [Epub ahead of print]1284 125303

A high-throughput LC-MS/MS assay for therapeutic drug monitoring of combined immunosuppressive and antimicrobial therapy.

Rongkun Zhu, Qiuyu Liao, Yuhong Gan, Yulin Peng, Guocui Zhang, Mengting Cai, Xiaobin Meng, Xuemin Guo, Huanghui Zeng, Caiyan Gan.

  This study established a rapid and reliable LC-MS/MS method for the simultaneous determination of six therapeutically relevant drugs: meropenem, vancomycin, voriconazole, linezolid, tacrolimus, and cyclosporine A. A key methodological advance is the first systematic characterization of the specific SRM transition of commercially available 13C₂,d₄-cyclosporine A, and its first application as an internal standard for this multiplex assay.
METHODS: The mass spectrometry parameters were optimized using a univariate approach to define the qualitative and quantitative ion transitions. Blood samples were extracted with methanol and quantified via the internal standard method, with calibration curves established for concurrent monitoring of the six target drugs. The developed method underwent comprehensive validation for selectivity, matrix effects, LLOQ, carryover, accuracy, precision, stability, dilution integrity, and calibration curve linearity. Its preliminary clinical applicability was assessed via pilot comparison with routine clinical reference methods: chemiluminescence immunoassay for immunosuppressants and conventional LC-MS/MS for antimicrobials.
RESULTS: The analytical method demonstrated excellent selectivity, with no detectable interfering peaks at the expected retention times for all analytes and internal standards. Matrix effects and carryover were minimal. In the accuracy test, both intra- and inter-batch accuracies ranged from 87.04% to 113.04%. The coefficients of variation (CV) for precision were within ±12.41%. Stability was satisfactory. The recoveries for all drugs were between 88.30% and 106.43%. Good linearity was demonstrated across the concentration ranges, with correlation coefficients (r) > 0.99. The method comparison agreement ranged from 86.47% to 111.53%.
CONCLUSION: The specific SRM transition of 13C₂,d₄-cyclosporine A was characterized for the first time, and a robust LC-MS/MS method was established for the simultaneous quantification of the six target drugs in human whole blood.

Keywords:  Cyclosporine A internal standard; Method validation; Preliminary clinical evaluation; Simultaneous quantification; Stable isotope-labeled internal standard; Therapeutic drug monitoring (TDM)

DOI:  https://doi.org/10.1016/j.jchromb.2026.125303
Ther Drug Monit. 2026 Sep 23.

Development, Validation, and Clinical Application of a Simultaneous Quantification Method of Seventeen Antituberculosis Drugs in Plasma, Serum, or Saliva Using LC-MS/MS.

Mathieu S Bolhuis, Erwin M Jongedijk, Onno W Akkerman, Daan J Touw, Marieke G G Sturkenboom.

   BACKGROUND: Tuberculosis (TB) remains a global health issue. Treatment involves multiple anti-TB drugs, and pharmacokinetic variability may lead to toxicity or ineffective treatment. Therapeutic drug monitoring may help address these challenges. This study aimed to develop a rapid method to analyze anti-TB drugs in plasma, serum, and saliva.
METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of anti-TB drugs in plasma, serum, and saliva. The method was validated according to European Medicines Agency and Food and Drug Administration guidelines, assessing linearity, selectivity, carry-over, accuracy, precision, dilution integrity, matrix effect, recovery, and stability. Performance was evaluated across plasma, serum, and saliva and in an external quality control program.
RESULTS: A method with a runtime of 1.7 minutes was developed to analyze 17 anti-TB drugs and 6 metabolites: (N-desmethyl-)bedaquiline, clofazimine, delamanid, ethambutol, ethionamide, gatifloxacin, (acetyl-)isoniazid, (nor)levofloxacin, linezolid, moxifloxacin, pretomanid, protionamide, pyrazinamide, (desacetyl-)rifabutin, (desacetyl-)rifampicin, (desacetyl-)rifapentine, and tedizolid. The method demonstrated linearity, selectivity, and minimal carry-over. Accuracy and precision were within acceptable limits for all drugs and metabolites, with the largest biases observed for rifabutin (-13.5%) and rifapentine (11.6%). Most drugs were stable, although delamanid and protionamide required immediate processing. All analytes met the precision criteria (CV <15%) in serum and saliva, whereas matrix-specific calibrators were required for some analytes to achieve acceptable accuracy (bias <15%).
CONCLUSIONS: This validated LC-MS/MS method allows simultaneous analysis of multiple anti-TB drugs in plasma, serum, and saliva, facilitating clinical and research applications. The method has been successfully applied in clinical practice.

Keywords:  LC-MS/MS; bioanalysis; multidrug-resistant tuberculosis; therapeutic drug monitoring; tuberculosis

DOI:  https://doi.org/10.1097/FTD.0000000000001532
J Anal Toxicol. 2026 Sep 22. pii: bkag086. [Epub ahead of print]

Development and Validation of an LC-MS/MS Method for the Detection of Substituted Phenethylamines and Phenidates Designated as Temporary Narcotics in Urine.

Sujin Jeong, Yoseob Kim, Seojin Kang, Chae Eun Lee, Moonhee Jang, Hyung Min Kim.

  Novel psychoactive phenethylamines and phenidates are synthetically designed analogs of conventional substances such as 3,4-methylenedioxymethamphetamine (MDMA), amphetamine, and methylphenidate, developed to circumvent existing drug regulations. In Korea, the abuse of these substances has increased, particularly among habitual users and newly addicted younger individuals. To address this issue, new substances are regulated through the analog legislation and the temporary narcotic designation system. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using solid-phase extraction (SPE) was developed and validated for the simultaneous determination of 13 substituted phenethylamines and 5 substituted phenidates in human urine. The target analytes included 5-MAPDB, BDB, 4-FEA, PMEA, 3-FPM, 25iP-NBOMe, 2C-N, 2C-TFM, 30C-NBOMe, 3C-P, 4-MMA-NBOMe, DOI, and 2C-H, as well as 4-MeTMP, 4F-EPH, 4F-MPH, IPPH, and HDEP-28. No interfering peaks were observed at the retention times of analytes and internal standards. Linearity was confirmed over analyte-specific concentration within the overall range of 0.1-100 ng/mL, with coefficient of determination (r2) exceeding 0.995. The limits of detection and quantification were 0.001-0.1 ng/mL and 0.1-0.2 ng/mL, respectively. Accuracy and precision, expressed as bias and coefficient of variation (CV), were within ±10% and <10%, respectively. The CVs for matrix effects and extraction efficiencies were approximately 10%, and the extraction efficiencies exceeded 70%, confirming the reliability of the method for forensic applications. The validated method enables simultaneous determination of substituted phenethylamines and phenidates in urine and facilitates prompt positive identification in forensic casework. In addition, the structural information, MRM conditions, and analytical method validation results presented in this study can serve as a basis for expanding research on substances with similar core structures, which is expected to strengthen regulatory responses to evolving drug trends.

Keywords:  LC–MS/MS; Method validation; Novel psychoactive substances; Substituted phenethylamines; Substituted phenidates

DOI:  https://doi.org/10.1093/jat/bkag086
Rapid Commun Mass Spectrom. 2026 Dec 15. 40(23): e70188

Preparation of Deuterium-Labeled Tadalafil and Omeprazole by Carbon Hydrogen-Deuterium Exchange: Hydrogen Scrambling, Acid-Induced Transformation, and LC-MS Applications.

Martyna Hofman-Kotyla, Paulina Grocholska-Zamiara, Robert Wieczorek, Remigiusz Bąchor.

   RATIONALE: Stable isotope-labeled compounds are indispensable internal standards for reliable liquid chromatography-mass spectrometry (LC-MS) analysis, but their preparation frequently requires multistep synthesis. Base-catalyzed hydrogen-deuterium exchange at carbon centers provides a simple alternative for generating stable isotopologues. Beyond quantitative applications, carbon-bound deuterium may serve as a mechanistic probe for investigating gas-phase fragmentation and chemically induced molecular transformations. In this work, tadalafil and omeprazole were selected as complementary model compounds to evaluate both the analytical applicability of deuterium-labeled standards and the behavior of carbon-bound deuterium during tandem mass spectrometry and acid-induced transformation.
METHODS: Deuterium-labeled tadalafil and omeprazole were prepared by base-catalyzed hydrogen-deuterium exchange under optimized reaction conditions. The obtained isotopologue mixtures were characterized using high-resolution ESI-MS, multistage tandem mass spectrometry, 1H NMR spectroscopy, and density functional theory (DFT) calculations. Their chromatographic behavior, short-term isotope stability, and proof-of-concept applicability as LC-MS internal standards were evaluated using chromatographic comparison and analysis of post-dose human urine samples.
RESULTS: Base-catalyzed hydrogen-deuterium exchange produced isotopologue mixtures dominated by tadalafil-d3 and omeprazole-d2. High-resolution mass spectrometry and 1H NMR spectroscopy consistently identified selective incorporation of carbon-bound deuterium, whereas DFT calculations explained the observed regioselectivity of the exchange. Tandem mass spectrometry of deuterated tadalafil revealed product-ion mass shifts consistent with hydrogen scrambling, demonstrating migration of carbon-bound deuterium during collision-induced dissociation and providing additional insight into the dissociation mechanism. In contrast, deuterium-labeled omeprazole retained its isotope label during acid-induced transformation. Both labeled compounds exhibited chromatographic behavior suitable for proof-of-concept LC-MS applications.
CONCLUSIONS: Base-catalyzed carbon hydrogen-deuterium exchange provides a rapid and cost-effective approach for preparing stable isotope-labeled tadalafil and omeprazole suitable for LC-MS applications. The study further demonstrates that carbon-bound deuterium constitutes a valuable mechanistic probe, enabling investigation of hydrogen scrambling during tandem mass spectrometry and evaluation of isotope-label stability during acid-induced molecular transformation. The combined HRMS, tandem MS, NMR, and DFT approach establishes a comprehensive framework for characterization of deuterium-labeled compounds and supports their future application in quantitative analysis as well as structural elucidation by mass spectrometry.

Keywords:  LC‐MS; hydrogen scrambling; hydrogen–deuterium exchange; isomerization under acidic conditions; isotope dilution

DOI:  https://doi.org/10.1002/rcm.70188
Anal Chim Acta. 2026 Nov 08. pii: S0003-2670(26)01062-7. [Epub ahead of print]1422 346112

Position specific 15N isotope analysis at natural abundance by 1H NMR.

Maylis Couton, Margot Sanchez, Valérie Fargeas, Illa Tea.

  Stable isotope analysis of nitrogen (N) at natural abundance provides key insights into biochemical processes. However, position-specific isotope analysis (PSIA) of 15N remains technically challenging due to the low sensitivity of conventional approaches. The approach we present here relies on resolving the 1H-15N doublets embedded within dominant 1H-14N triplets and quantifying them via spectral deconvolution under rigorously optimized quantitative NMR conditions. Using ammonium chloride as a model, we established the experimental and processing parameters required for precision (≤2‰) and trueness (<3‰), validated against isotope ratio monitoring Mass Spectrometry (irm-MS). The method was successfully extended to structurally more complex compounds (aniline, indole, urea, and N-acetylated tryptophan methyl ester), achieving accurate position-specific δ15N determinations. Importantly, we demonstrate the feasibility of applying this strategy to a human urine sample, enabling direct δ15N measurement of endogenous urea in a complex biological matrix at natural abundance. This non-destructive method requires minimal sample amounts and offers a robust complement to irm-MS, expanding the analytical toolbox for nitrogen isotope research in environmental, biochemical, and biomedical contexts.

Keywords:  (1)H NMR; Natural abundance; Nitrogen isotope; Position specific isotope analysis

DOI:  https://doi.org/10.1016/j.aca.2026.346112
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Sep 19. pii: S1570-0232(26)00393-4. [Epub ahead of print]1284 125304

Optimization of untargeted lipidomics analysis of human plasma after Dangguijakyak-san administration.

Jin Ju Park, Sook Jin Seong, Jae Hwa Lee, Ji Seo Park, Hae Won Lee, Young-Ran Yoon, Soo-Jin Park, Mi-Ri Gwon.

  Untargeted lipidomics enables comprehensive characterization of lipid profiles; however, analytical variability in sample preparation, chromatographic conditions, and data processing often limits reproducibility. Thus, this study aimed to optimize untargeted lipidomics analysis by evaluating chromatographic columns, mobile-phase additives, and lipid extraction methods to maximize feature detection, reproducibility, and overall data quality. Chromatographic performance was compared between ethylene-bridged hybrid (BEH) and charged-surface hybrid columns. For mobile-phase additives, 10 mM ammonium formate (AmFa) with or without 0.1% formic acid (Fa) was evaluated in positive ionization mode, whereas 10 mM ammonium acetate (AmAc) with or without 0.1% acetic acid was evaluated in negative ionization mode. Lipid extraction was assessed using chloroform-, methyl tert-butyl ether-, or butanol-based methods, with or without re-extraction. The final optimized workflow consisted of a BEH column, 10 mM AmFa with 0.1% Fa in positive mode, 10 mM AmAc in negative mode, and the Folch extraction method. The optimal analytics applied to plasma samples collected from women with primary dysmenorrhea, before and after Dangguijakyak-San (DJS) administration, identifying 321 lipid species across major classes, including glycerophospholipids, glycerolipids, and sphingolipids. Principal component analysis of quality control samples demonstrated tight clustering, and 14 internal standards exhibited relative standard deviations below 30%, confirming analytical robustness. Four lipid species were significantly altered: three phosphatidylcholines decreased, and one ceramide phosphoinositol increased after DJS administration. These alterations suggest modulation of oxidative stress-related lipid pathways and sphingolipid metabolism. This optimized analytical workflow provides reproducible plasma lipidomics and has potential applications in studies of traditional herbal medicines.

Keywords:  Analytics optimization; Human plasma; UHPLC–QTOF/MS; Untargeted lipidomics

DOI:  https://doi.org/10.1016/j.jchromb.2026.125304
Toxics. 2026 Aug 26. pii: 763. [Epub ahead of print]14(9):

Determination of 25 Organophosphate Ester Flame Retardants in Soils by Accelerated Solvent Extraction-Ultra-High Performance Liquid Chromatography.

Ban Cao, Yinjun Shi, Zuguo Hu, Mingli Ye.

  Organophosphate esters (OPEs) are commonly used flame retardants and plasticizers, which can easily be released into the soil environment and have potential hazards such as neurotoxicity and developmental toxicity. Establishing an efficient and sensitive detection method plays a crucial role in soil pollution assessment. Currently, there have been numerous studies on the detection of OPEs in soil using ultrasonic extraction and solid-phase extraction, while relatively few studies have focused on the analysis of multiple OPEs in soil by accelerated solvent extraction combined with d-SPE clean-up and ultra-high performance liquid chromatography-tandem mass spectrometry. The d-SPE purification method eliminates the need for column passage, shortens sample preparation time, and avoids the risk of background contamination from OPEs that may be introduced by SPE. This study developed a liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) method for the determination of 25 OPEs in soil, and systematically optimized the pretreatment and instrumental analysis conditions. Accelerated solvent extraction was used for pretreatment, and dichloromethane-methanol (1:1, V/V) was determined as the optimal extraction solvent. A mixed adsorbent of N-propyl ethylenediamine (PSA) and C18 was selected for dispersive purification, effectively removing matrix interference and improving recovery rates. The mass spectrometry parameters such as collision energy and declustering voltage were optimized, significantly enhancing ion response intensity and detection sensitivity. The method showed good linearity within the concentration range of 1-100 ng/mL, with correlation coefficients all greater than 0.994. The spiked recovery rates ranged from 70.6% to 111% with a relative standard deviation (RSD) of 1.2-11.8%. The precision and accuracy met the requirements for environmental sample analysis. The method was applied to the detection of actual soil samples, and the results were stable and reliable. This method is simple to operate, highly sensitive, and widely applicable, providing reliable technical support for the pollution monitoring, source tracing, and ecological risk assessment of OPEs in soil.

Keywords:  accelerated solvent extraction; liquid chromatography-tandem mass spectrometry; organophosphate esters (OPEs); soil

DOI:  https://doi.org/10.3390/toxics14090763
Toxins (Basel). 2026 Sep 14. pii: 395. [Epub ahead of print]18(9):

Multiclass Cyanotoxins in Aquatic Environments: Occurrence, Impacts and Detection Techniques.

Galina Nugumanova.

  Cyanobacterial harmful algal blooms (cyanoHABs) typically involve the release of a wide spectrum of toxic metabolites produced by different cyanobacteria, which can cause adverse health effects in animals and humans. At the same time, many cyanobacteria are capable of producing more than one toxin simultaneously. As a result, the multiclass toxins released into aquatic environments can exert complex and combined effects on various ecosystem components. This review aims to provide an overview of the occurrence and effects of different cyanotoxins in aquatic systems and current analytical methods of their simultaneous detection. The main classes of cyanotoxins have been considered in this review. The occurrences, properties, and environmental and health impacts of these toxins are described. The main methods for cyanobacterial toxin analysis are covered. A comprehensive review and analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection and sample preparation approaches used to identify various classes of cyanotoxins is provided, together with a practical decision tree for analytical workflow selection according to toxin class and sample matrix, highlighting key trends and outlining directions for further research in this area.

Keywords:  BMAA; LC-MS/MS; anatoxin-a; cyanoHABs; cyanotoxins; cylindrospermopsin; microcystin; multiclass analysis; nodularin; saxitoxin

DOI:  https://doi.org/10.3390/toxins18090395
Front Vet Sci. 2026 ;13 1951616

A complementary dual-platform analytical strategy for resolving matrix effects and stability limitations in bioanalysis.

Jang Mi Han, Jungho Yoon, Young Beom Kwak.

   Introduction: Famotidine and omeprazole are widely used for the treatment of acid-related disorders in racehorses, and their combined administration is expected to provide complementary therapeutic effects due to their distinct mechanisms of action. However, their markedly different physicochemical properties present substantial challenges for simultaneous quantification in biological matrices. This study aimed to develop and validate complementary analytical strategies for the simultaneous determination of famotidine and omeprazole in equine plasma using high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
Methods: Dual-platform analytical methods were developed, with sample preparation conditions tailored to the physicochemical properties of each compound. For HPLC analysis, the effects of sample preparation conditions on the chromatographic behavior and linearity of famotidine and omeprazole were evaluated. For UHPLC-MS/MS analysis, a pH-neutral sample preparation strategy was established to preserve the stability of acid-labile omeprazole. The developed methods were validated according to relevant analytical validation parameters and subsequently applied to equine plasma samples collected following drug administration for pharmacokinetic evaluation.
Results: In HPLC analysis, famotidine exhibited pH-dependent peak splitting associated with changes in its ionization state, which was resolved by acidifying the sample preparation solvent. Omeprazole showed limited solubility in aqueous environments, resulting in nonlinearity at higher concentrations; this was overcome by acidifying the plasma matrix, enabling quantification over 100-5,000 μg/mL. Although the HPLC method effectively characterized the analytical behavior of both compounds, its sensitivity was insufficient for trace-level pharmacokinetic analysis. Therefore, a UHPLC-MS/MS method was developed using pH-neutral sample preparation, enabling the simultaneous determination of omeprazole and its metabolite, omeprazole sulfone. The UHPLC-MS/MS method achieved an LLOQ of 50 ng/mL, representing an approximately 2,000-fold improvement in analytical sensitivity compared with HPLC. The developed methods demonstrated acceptable validation performance and were successfully applied to equine plasma samples, enabling the determination of concentration-time profiles and key pharmacokinetic parameters.
Discussion: The findings demonstrate that differences in the physicochemical properties and stability of famotidine and omeprazole require platform-specific sample preparation strategies for reliable simultaneous quantification. The complementary use of HPLC and UHPLC-MS/MS provided both characterization of compound-specific analytical behavior and sensitive quantification of trace drug concentrations in equine plasma. This analytical framework may support pharmacokinetic studies of famotidine and omeprazole in equine models.

Keywords:  HPLC; UHPLC-MS/MS; equine plasma; famotidine; omeprazole; omeprazole sulfone; pharmacokinetics

DOI:  https://doi.org/10.3389/fvets.2026.1951616
Front Nutr. 2026 ;13 1883491

Plant-based dietary biomarkers: from discovery to validation using mass spectrometry-based metabolomics.

Gerben de Gier, Michiel G J Balvers, Edith J M Feskens, Renger F Witkamp, Eva Jermendi.

  As diets shift globally toward plant-based foods, objective tools are needed to monitor intake and evaluate the resulting health outcomes. Biomarkers of dietary exposure offer complementary objective measurements for self-reported dietary assessment methods. Recent developments of mass spectrometry-based metabolomics have enabled the discovery of many emerging biomarkers for plant-based foods; yet a substantial gap remains between discovery and clinical application. This narrative review addresses the question: which plant-based food biomarkers are sufficiently validated for use in dietary exposure assessments, and which analytical and biological obstacles currently limit their application in population studies? Biomarkers reported in the literature were categorized as validated, emerging, or discovery-stage candidates. Analytical methodologies for biomarker quantification are also discussed, including high-resolution mass spectrometry platforms, and emerging techniques. Challenges identified in biomarker validation involve inter-individual metabolic variability, inadequate food specificity, and matrix effects in biological samples. Integration of foodomics databases with targeted validation studies potentially offers an efficient approach to speed up biomarker discovery. Multi-marker panels combining structurally different metabolites are promising for assessing complex plant-based dietary patterns.

Keywords:  dietary biomarkers; foodomics; mass spectrometry; metabolomics; plant-based foods

DOI:  https://doi.org/10.3389/fnut.2026.1883491
Biomed Chromatogr. 2026 Nov;40(11): e70625

Quantification of Amlexanox in Beagle Dog Plasma by LC-MS/MS and Its Application in a Comparative Pharmacokinetic Study of Conventional and Sustained-Release Tablets.

Yanmei Yi, Wei He, Xiaochen Niu, Jianchun Zhao.

  Amlexanox, a 5H-benzopyranopyridine derivative with emerging therapeutic potential in metabolic and inflammatory diseases, lacks a validated analytical method for pharmacokinetic studies in beagle dogs. A sensitive LC-MS/MS method was developed and validated for amlexanox in beagle dog plasma according to ICH M10 guidelines. Propranolol served as internal standard. Samples were processed by protein precipitation, and separation was achieved on a CAPCELL PAK C18 column using gradient elution with water and acetonitrile (both containing 0.2% formic acid) at 400 μL/min. Detection used positive electrospray ionization with MRM of m/z 299.0 → 281.0 for amlexanox and m/z 260.0 → 116.1 for IS. The method showed good linearity over 40-2000 ng/mL (R2 > 0.99). All validation parameters met acceptance criteria. The method was applied to a three-period fixed-sequence study in beagle dogs (n = 3, male) comparing intravenous injection (1 mg/kg), conventional tablets (75 mg), and sustained-release tablets (150 mg). The sustained-release formulation prolonged Tmax (3.67 vs. 1.00 h) and residence time (two-fold increase in t1/2 and MRT), and improved absolute bioavailability from 25.36% to 33.09% (relative bioavailability: 151.30%). These findings support the clinical development of a sustained-release amlexanox formulation with reduced dosing frequency and improved patient compliance.

Keywords:  LC–MS/MS; amlexanox; beagle dog; pharmacokinetics; sustained‐release tablet

DOI:  https://doi.org/10.1002/bmc.70625
Toxics. 2026 Aug 26. pii: 753. [Epub ahead of print]14(9):

Per- and Polyfluoroalkyl Substances (PFAS) in Michigan: A Novel High-Throughput Method with Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis of 39 PFAS Compounds in Human and Bovine Dried Blood Spots (DBS).

Jessica M Morrison, Sarah Y Lockwood-O'Brien, Chelsea A Bielicki, Douglas J Carmack, Julia R Feeley, Timothy A Karrer, Matthew J Geiger.

  This study presents a novel method for analyzing per- and polyfluoroalkyl substances (PFAS) in dried blood spots (DBS), employing a hybrid solvent-based and matrix-matched calibration curve, minimal sample volume, and high-throughput preparation. This process produces a concentrated, purified sample, which is analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). As part of method validation, key performance metrics such as accuracy, precision, sensitivity, carryover, specificity, interferences, and maximum dilution capability were assessed. The method quantifies 39 PFAS compounds across various classes, achieving limits of detection in the low ng L-1 range, with most analytes having a limit of quantitation below the first calibrator (50 ng L-1). The total method accuracy exceeded 81%, surpassing the performance goal of ≥70%, while method imprecision was below 28%, outperforming the goal of ≤30%. Specificity, as measured by a normalized percent matrix bias, was below 11%, with limited and minimal matrix effects observed. Interference from bile acids was determined to be insignificant at biologically relevant concentrations. Over the past three years, this method has been successfully utilized in biomonitoring studies of Michigan residents, demonstrating its robustness and efficiency.

Keywords:  analytical sample preparation methods; biological monitoring; dried blood spots; environmental monitoring; fluorocarbons; liquid chromatography–mass spectrometry

DOI:  https://doi.org/10.3390/toxics14090753
Metabolites. 2026 Aug 24. pii: 604. [Epub ahead of print]16(9):

Metabodeconplus-An R Package for Automated Deconvolution and Alignment of 1D NMR Metabolomics Data.

GCKD Investigators

  Background: In one-dimensional NMR spectra of complex biofluids such as urine and plasma, extensive signal overlap obscures individual metabolite signals. Resolving this overlap by deconvolution is only the first step: turning a set of spectra into a table for subsequent statistical analysis also requires the alignment of signals across samples and their integration into a single feature matrix. Methods: Here, metabodeconplus is presented, an R package that unifies this entire path into a single reproducible end-to-end workflow. From raw one-dimensional spectra, it deconvolutes overlapping signals, aligns resulting signals across samples, and integrates them into a data matrix for built-in sample classification or downstream statistical analysis. Automated parameter optimization removes manual tuning, and a Rust computational backend with parallelization leads to fast runtimes. Results: On the simulated Sim3 spectra, a combined score of correctly identified signals and reconstruction accuracy (maximum 1) rose from 0.712 for the predecessor package to 0.801 for metabodeconplus. For the urinary AKI dataset, metabodeconplus reached a classification accuracy of 73.7 ± 2.20% and an AUC=0.827±0.025, which is comparable to the binning baseline. An advantage is the potential unambiguous metabolite assignment of the deconvoluted signals. Conclusions: The package is freely available as open source on GitHub and on CRAN.

Keywords:  1D NMR; R package; deconvolution; metabolites; quantification; signal identification

DOI:  https://doi.org/10.3390/metabo16090604
Metabolomics. 2026 Sep 25. pii: 161. [Epub ahead of print]22(5):

Systematic mass-spectrometry-guided metabolic fingerprinting elucidates diversity of specialized metabolites across the Brassicaceae.

Felicia C Wolters, Tina Woldu, M Eric Schranz, Marnix H Medema, Klaas Bouwmeester, Justin J J van der Hooft.

   BACKGROUND: Plants produce diverse bouquets of specialized metabolites (SMs), yet only a fraction of the vast phytochemical space hasbeen explored to date.Comparative analysis of SM profiles can reveal hotspots of biochemical novelty, while systematic profilingacross taxonomic levels does presently not cover large plant families.
MATERIALS AND METHODS: To study core and accessory SM profiles in the Brassicaceae plant family, we fingerprinted 14 species by Liquid-ChromatographyMass-Spectrometry (LC-MS/MS). We develop standardized experimental and computational workflows integrating in silicoannotation tools to study consensus compound class and substructure distributions of SMs. Furthermore, we investigate thecongruence of chemotaxonomy and species phylogeny across an extended panel of 17 species.
RESULTS: Unique metabolite profiles were outstanding in Camelina sativa, Capsella rubella, and Barbarea vulgaris, with the largest uniqueterpenoid profile annotated in C. sativa, accounting for 33.5% and 55.6% in positive and negative ionization mode, respectively.Substructure motifs were found to overlap with compound class predictions, highlighted for triterpenoids in Camelinodae. Furthermore, dual-tissue chemotaxonomic clustering resembled relationships of Brassica nigra, B. oleracea, B. carinata, and B. juncea, and corresponding B and C subgenomes across tissues.
CONCLUSION: We anticipate that our systematic approach can serve as a blueprint for investigating biochemical diversity in other plant lineagesand can boost the characterization of plant natural product pathways.

Keywords:   in silico annotation; Brassicaceae; Chemotaxonomy; Computational metabolomics; Metabolic fingerprinting; Specialized metabolites; Terpenes

DOI:  https://doi.org/10.1007/s11306-026-02537-y
Nutrients. 2026 Sep 11. pii: 2986. [Epub ahead of print]18(18):

Comprehensive Comparison of Three Automated 25-Hydroxyvitamin D Immunoassays with a VDSCP Traceable LC-MS/MS Method Across Clinically Relevant Decision Thresholds.

Szilvia Racz, Luca Jozsa, Amrit Pal Bhattoa-Buzas, Emese Szmik, Edit Kalina, Sandor Barath, Dora Bencze, Etienne Cavalier, Harjit Pal Bhattoa.

   BACKGROUND/OBJECTIVE: Accurate measurement of serum 25-hydroxyvitamin D (25OHD) is essential for the diagnosis and management of vitamin D deficiency. Although automated immunoassays are widely used in routine clinical practice, analytical variability between platforms may lead to inconsistent patient classification around clinically relevant decision thresholds. This study comprehensively compared three automated immunoassays (DiaSorin Liaison XL, Beckman Coulter DxI 9000, and Roche cobas e602) with a Vitamin D Standardization Certification Program (VDSCP)-traceable liquid chromatography-tandem mass spectrometry (LC-MS/MS) reference method.
METHODS: A total of 400 anonymized adult outpatient serum samples were prospectively collected and equally distributed across four predefined vitamin D concentration categories (<25, 25-49, 50-74, and ≥75 nmol/L). Samples were stored at -70 °C before simultaneous analysis on the three automated immunoassays and the reference LC-MS/MS platform. Method comparison included Deming regression, Bland-Altman analysis, and evaluation of diagnostic performance at clinically relevant thresholds (<25, <50, and <75 nmol/L). The effects of sample storage, vitamin D metabolites (C3-epi-25OHD3 and 24,25-dihydroxyvitamin D3), and external quality assessment (DEQAS) performance were also investigated.
RESULTS: Significant analytical differences were observed between all immunoassays and LC-MS/MS. Roche cobas e602 consistently overestimated 25OHD concentrations, resulting in reduced sensitivity but excellent specificity and positive predictive value across all clinical thresholds. Beckman Coulter demonstrated the highest sensitivity for detecting vitamin D deficiency but generated more false-positive classifications, particularly at the <75 nmol/L threshold. DiaSorin Liaison XL achieved the best overall balance between sensitivity, specificity, accuracy, and agreement with LC-MS/MS, with Cohen's κ values ranging from 0.818 to 0.895 across decision thresholds. Higher concentrations of C3-epi-25OHD3 and 24,25-dihydroxyvitamin D3 were associated with greater positive bias in Roche measurements, while Beckman Coulter showed no significant metabolite-related interference, although the present correlation analyses could not establish independent analytical interference. DEQAS performance did not consistently reflect agreement with LC-MS/MS in patient samples.
CONCLUSIONS: Despite ongoing international standardization efforts, clinically important differences remain among automated 25OHD immunoassays. Assay-specific analytical characteristics significantly influence patient classification around established decision thresholds. DiaSorin Liaison XL demonstrated the closest overall agreement with the reference LC-MS/MS method, whereas Beckman Coulter prioritized sensitivity and Roche specificity. These findings highlight the importance of method-aware interpretation of vitamin D results and support continued assay harmonization to improve comparability in routine clinical practice.

Keywords:  25-hydroxyvitamin D; Beckman Coulter DxI 9000; DiaSorin Liaison XL; LC-MS/MS; Roche Cobas e602; comparison study; vitamin D metabolites

DOI:  https://doi.org/10.3390/nu18182986
Metabolites. 2026 Aug 31. pii: 633. [Epub ahead of print]16(9):

Organ-Resolved Untargeted LC-MS/MS Profiling of Putative Metabolite Families in Aerial Organs of Vitex negundo L.

Menghu Wang, Xinyang Wang, Xue Jiang, Yafeng Zuo, Dong Liu, Qianqian Jiang, Xiangsong Meng.

   BACKGROUND/OBJECTIVES: Vitex negundo L. is a multi-part medicinal and aromatic plant, but organ-resolved chemical information for its aerial organs remains incomplete. This study generated an exploratory paired untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) dataset for flowers, stems, and leaves and prioritized organ-associated putative metabolite families for subsequent validation.
METHODS: To reduce inter-individual background variation, flower, stem, and leaf tissues were harvested from the same three V. negundo individuals to form n = 3 paired plant blocks. LC-MS/MS feature data were filtered using pooled quality-control relative standard deviation (QC RSD ≤ 30%), median-normalized and log2-transformed. Principal component analysis (PCA) provided an unsupervised overview, and paired feature-level comparisons were performed using paired t-tests with Benjamini-Hochberg false discovery rate (FDR) correction.
RESULTS: A total of 42,125 QC-filtered LC-MS features were retained. PCA showed organ-associated separation among flowers, stems, and leaves. Using FDR < 0.05 and |mean paired log2 difference| ≥ 1, paired screening yielded 13,043, 22,836, and 13,393 FDR-supported LC-MS features for flower vs. stem, flower vs. leaf, and stem vs. leaf comparisons, respectively. Higher-scoring putative annotations suggested organ-associated putative family-level patterns, including flavonoid-related, hydroxycinnamic-acid derivative, phenolamide-related, triterpenoid-related, iridoid-related, and lignan-related signals.
CONCLUSIONS: This organ-resolved dataset provides a chemical-feature framework for V. negundo aerial organs and putative family-level patterns for future structural confirmation, targeted quantification, bioactivity evaluation, and quality-marker development. Feature counts are not equivalent to unique metabolite counts, and compound labels require validation before being interpreted as confirmed structures.

Keywords:  LC-MS/MS; Verbenaceae; Vitex negundo; medicinal-resource evaluation; organ-resolved profiling; plant specialized metabolism; putative annotation; putative metabolite families; untargeted metabolomics

DOI:  https://doi.org/10.3390/metabo16090633
J Chromatogr A. 2026 Sep 10. pii: S0021-9673(26)00776-4. [Epub ahead of print]1787 467450

Hyphenation of supercritical fluid chromatography to high resolution Orbitrap mass spectrometry for the purpose of non-targeted small molecule analysis.

Mikaela J Radke, Alan White, Frederic D L Leusch, Sarah L Cresswell.

  To cater for the rising need to identify current and emerging environmental contaminants, a hybrid chromatography-mass spectrometry interface was developed for supercritical fluid chromatography. Supercritical fluid chromatography was selected for normal phase and reverse phase selectivity and was hyphenated to an Orbitrap mass analyser for application to non-targeted methods and high-resolution mass spectrometry. This interface was achieved through ion source parameter optimisation and the use of make-up solvent flow delivered by a sheath pump. The method presented here was developed based on 23 reference standards of environmental contaminants, pharmaceuticals and polar compounds which have logD (pH 5.5) values between -2.50 and 4.96. Further non-targeted method evaluation was performed on treated wastewater effluent samples, where this non-targeted methodology allowed for the identification of 88 compounds across both positive and negative ion modes. Chromatography separation characteristics were assessed across both reference standard and WWTP sample runs. The major separation characteristic was polarisability represented by acidic and basic hydrogen bonding. Compounds with a pKa of <5 eluted earlier in the run and compounds with a pKa of 8-13 eluted later due to the acidity of the mobile phase and the capability of the Acquity Torus DIOL column for acidic hydrogen bonding. These separation characteristics demonstrate that this method has wide application to a large number of functional groups, with orthogonal separation capability for polar compounds to support reverse phase liquid chromatography methods.

Keywords:  Environmental contaminants; High resolution mass spectrometry; Non-targeted analysis; Supercritical fluid chromatography; Wastewater

DOI:  https://doi.org/10.1016/j.chroma.2026.467450
Anal Chim Acta. 2026 Nov 08. pii: S0003-2670(26)01078-0. [Epub ahead of print]1422 346128

Instant CMQT stabilization coupled UHPLC-MS/MS for the quantification of reduced and total forms of cysteine, homocysteine, and glutathione in blood plasma.

Yongyin Zhou, Haizhu Tan, Yingtong Chen, Hongjun Luo, Hui Li, Wenhong Luo, Yingxiu Xiao, Zhexuan Lin.

   BACKGROUND: Reduced and total forms of cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are key players in redox homeostasis, and their dysregulation is linked to diseases such as sepsis. However, accurate quantification of reduced thiols in plasma remains challenging due to rapid air oxidation of native thiols. In this work, a thiol-specific reagent (2-chloro-1-methylquinolinium tetrafluoroborate, CMQT) that reacts rapidly under mild conditions was coupled with UHPLC-MS/MS to develop a method for the simultaneous determination of reduced and total forms of Cys, Hcy, and GSH in plasma samples.
RESULTS: CMQT rapidly stabilizes native thiols in whole blood without inducing hemolysis. The proposed method was validated for linearity, precision, accuracy and sensitivity. Calibration curves showed excellent linearity (R2 > 0.9993). Intra- and inter-day precisions ranged from 0.14% to 7.23%, and accuracy from -5.62% to 4.15%. The limits of detection for the reduced and total forms of Cys, Hcy and GSH ranged from 0.051 to 0.88 μM. The method was successfully applied to a lipopolysaccharide-induced endotoxemia mouse model, revealing that reduced Hcy and reduced GSH may serve as potential biomarkers for early and late oxidative stress, respectively. The method was also validated on human plasma samples, confirming its applicability across species and revealing sex-related thiol differences.
SIGNIFICANCE: The combination of rapid and hemolysis-free thiol stabilization with MS-based structural characterization provides a robust analytical tool for thiol redox studies and could be extended to other biological matrices.

Keywords:  Cysteine; Endotoxemia; Glutathione; Homocysteine; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.aca.2026.346128
Molecules. 2026 Sep 09. pii: 3167. [Epub ahead of print]31(18):

Assembling an Expanded Metabolome for Camu-Camu (Myrciaria Dubia): Bioactive and Nutritional Compounds Relevant to Functional Food Research.

Subramanyam Ragupathy, Varathan Vinayagam, Velautham Saravanan, Arunachalam Thirugnanasambandam, Steven G Newmaster.

  Camu-camu (Myrciaria dubia) is an Amazonian shrub associated with riverine ecosystems and is recognized for its high vitamin C content and a broad range of nutritional and specialized metabolites. Previous studies have focused on specific metabolites or bioactive metabolites associated with health issues. Therefore, a comprehensive metabolomic survey using multiple analytical methods islacking. To address this gap in the scientific literature, we prepared a focused metabolomic inventory by integrating curated literature records with proton nuclear magnetic resonance (1H NMR) and liquid chromatography-mass spectrometry (LC-MS) observations from berry materials, including pulp, skin/peel, and seeds. Ourstudy added 107 metabolite annotation records that were not in the literature. The integration of the literature-derived and ourdatasets yielded a combined inventory of 260 metabolites. Seven solvents were compared using 1H NMR spectroscopy, and CH3OH:CD3OD (9:1, v/v) provided the broadest assigned coverage. LC-MS expanded the detection of lower-abundance and structurally diverse features, particularly phospholipids, quaternary ammonium compounds, organic acids, amino acid derivatives, and related metabolites. Compound-level and class-level assignments were distinguished, and interpretations were limited to the confidence supported by the available analytical evidence. The resulting inventory provides an expanded foundation for camu-camu quality evaluation, solvent selection, clinical trials, and targeted functional food research.

Keywords:  1H NMR; LC-MS; Myrciaria dubia; camu-camu; functional foods; metabolomics; phytochemicals; solvent extraction

DOI:  https://doi.org/10.3390/molecules31183167
Metabolomics. 2026 Sep 23. pii: 159. [Epub ahead of print]22(5):

Comparative 1H-NMR profiling of plasma and serum metabolites in giraffes and white rhinoceroses.

G Osthoff, S Mason, L Schmidt, A Tordiffe, F Deacon.

   BACKGROUND: Blood metabolite profiling can provide descriptive information on physiological variation in wildlife, but interpretation is sensitive to sampling design, specimen matrix and analytical batch.
OBJECTIVES: To characterise quantified blood metabolites in apparently healthy giraffes and white rhinoceroses, examine sex-associated differences within each species, and conduct an exploratory matrix-matched interspecies comparison.
METHODS: Proton nuclear magnetic resonance spectra were available for 24 valid giraffe sampling occasions from 19 animals and for 45 white rhinoceroses. Metabolites were quantified jointly from the original spectra. Design-defined comparisons used log₂-transformed concentrations, Welch tests, Benjamini-Hochberg false-discovery correction, effect sizes and permutation, rank-based, leave-one-out and annotation-confidence sensitivity analyses.
RESULTS: No sex-associated metabolite differences were detected among the 2017 Rooipoort giraffe serum samples. Nine metabolites met the primary false-discovery criterion in the rhinoceros sex comparison, although only N-acetylglucosamine showed consistently strong evidence across all sensitivity analyses. In the comparison of six verified giraffe and 45 rhinoceros EDTA-plasma samples, 36 of 50 metabolites differed by the primary analysis; 28 formed a conservative core supported by all statistical tests and all leave-one-giraffe-out analyses. The core remained significant after adjustment for sex. Collection year was completely confounded with serum versus plasma in giraffes and was not tested inferentially.
CONCLUSIONS: The two study groups had markedly different measured plasma metabolite profiles, with a coherent difference among branched-chain amino-acid-related metabolites. However, species was inseparable from NMR acquisition batch, and the small giraffe group limits biological and pathway-level inference. The concentrations provide exploratory baseline data rather than formal reference intervals.

Keywords:  EDTA plasma; Giraffe; Metabolomics; Nuclear magnetic resonance; Serum; White rhinoceros

DOI:  https://doi.org/10.1007/s11306-026-02535-0
Pharmaceuticals (Basel). 2026 Sep 03. pii: 1397. [Epub ahead of print]19(9):

Validated LC-MS/MS Methods for Quantification of AC02 and Porcine ACTH1-39 in Human Plasma: Application to a Phase I Study.

Shunbo Zhao, Bingda Wu, Hui Shen, Qi Zhou, Chang Shu, Li Ding.

  Background/Objectives: AC02 is a novel 39-amino-acid adrenocorticotropic hormone (ACTH) analogue designed for the treatment of infantile spasms. To support its clinical study, in which porcine ACTH1-39 served as the positive-control drug, reliable methods for the determination of AC02 and porcine ACTH1-39 in human plasma were required. Reported analytical methods for ACTH analogues are mainly immunoassays, which are easily affected by cross-reaction and the hook effect, necessitating a more selective analytical approach. Methods: Two LC-MS/MS methods were developed for the determination of ACTH analogues AC02 and porcine ACTH1-39 in human plasma. Human ACTH1-39 was included as a selectivity marker to confirm that endogenous ACTH does not interfere with the quantification of AC02. Based on the distinct concentration requirements and matrix challenges, two sample-preparation procedures were established: micro-solid-phase extraction coupled with protein precipitation for porcine ACTH1-39 (LLOQ 0.100 ng/mL), and acid-mediated protein precipitation for AC02 (LLOQ 0.500 ng/mL). The [M+6H]6+ ions were selected as precursor ions, and the corresponding 5+ fragment ions, formed by loss of the C-terminal phenylalanine, were used for MRM detection. Results: Despite a mass difference of only 0.98 Da between AC02 and human ACTH1-39, which are indistinguishable by mass spectrometry, baseline chromatographic separation was achieved. Both methods were fully validated in accordance with current bioanalytical guidelines. Conclusions: The validated methods were successfully applied to the phase I clinical study of AC02 and porcine ACTH1-39, enabling reliable quantification of the drug candidate and its active comparator, porcine ACTH1-39.

Keywords:  AC02 peptide; LC–MS/MS method; adrenocorticotropic hormone; micro-solid-phase extraction; pharmacokinetics

DOI:  https://doi.org/10.3390/ph19091397
Metabolites. 2026 Sep 07. pii: 654. [Epub ahead of print]16(9):

Chemical Derivatization Strategies for Expanding Small Biomolecule Analysis by MALDI-MS.

Xintong Hu, Qinxue Wang, Yingying Lin, Jingjing Wan.

  Small biomolecules provide important information on cellular metabolism, signaling and disease-associated molecular changes. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) enables rapid, high-throughput and spatially resolved molecular analysis, but its application to small biomolecules is often limited by poor ionization, low-mass background interference, ion suppression and insufficient structural information. To address these analytical challenges, this review summarizes chemical derivatization strategies that improve MALDI-MS analysis of small biomolecules. The representative strategies are discussed according to reaction mode and target functional group, including solution-phase, on-target, on-tissue, reactive-matrix-assisted and photo-/in-source approaches, with emphasis on amine-, carbonyl-, carboxyl- and double-bond-containing biomolecules. Chemical derivatization improves MALDI-MS performance by selectively modifying target functional groups, introducing charged or ionizable tags, enhancing molecular discrimination and providing additional structural information. These strategies have expanded the detection and annotation of neuroactive amines, carbonyl compounds, glycans, carboxylic acid metabolites and lipid double-bond isomers, while also extending MALDI-MS analysis to other specific functional groups, natural products and drug-related molecules. Overall, chemical derivatization is an important approach for improving the sensitivity, selectivity, structural annotation and analytical coverage of MALDI-MS-based small biomolecule analysis. Future developments should further address reaction selectivity, spatial fidelity, quantitative reliability and confident identification of derivatization products.

Keywords:  MALDI-MS; biomolecules; chemical derivatization; spatial metabolomics

DOI:  https://doi.org/10.3390/metabo16090654
Nat Protoc. 2026 Sep 21.

Tissue expansion mass spectrometry imaging (TEMI) for high-spatial-resolution multiomics molecular mapping.

Hua Zhang, Lang Ding, Thao Duong, Kendra G Selby, Wan-Jo Lee, Amy Hu, Haiyan Lu, Paul W Tillberg, Meng Wang, Lingjun Li.

High-spatial-resolution in situ mapping of biomolecules within tissue reveals critical insights into the complex molecular landscape and spatial organization of biological systems. Mass spectrometry imaging (MSI) is a powerful tool for spatially resolved molecular analysis of biological samples, with ongoing demand for improved spatial resolution. Tissue expansion combined with MSI (TEMI) is a recently developed approach that enables multiomics molecular mapping across various biological tissues with significantly improved spatial resolution. Unlike conventional methods that depend on instrument-based enhancements in spatial resolution, TEMI physically enlarges tissue samples via harsh-condition-free hydrogel expansion, achieving more than 3.5-fold increase in effective imaging resolution using standard MSI instrumentation. TEMI delivers single-cell spatial resolution in tissue samples and enables detection of biomolecular heterogeneity that remains uncharacterizable in unexpanded tissue using conventional MSI. Notably, TEMI supports high-spatial-resolution mapping of multiple biomolecular classes-including lipids, metabolites, N-glycans, peptides and proteins-within a single tissue sample. Here, we provide a detailed, step-by-step guide for TEMI, including hydrogel-based tissue expansion under mild conditions, cryosectioning of the expanded tissue-hydrogel sample, a comprehensive experimental workflow for multiomics TEMI on a single tissue section, data acquisition and visualization pipelines, as well as troubleshooting tips. Overall, we demonstrate that TEMI overcomes the long-standing spatial limitations of MSI without requiring hardware modifications, ensuring compatibility with existing MSI instruments and promoting broad accessibility and adoption within the research community.

DOI: https://doi.org/10.1038/s41596-026-01427-w
Metabolites. 2026 Aug 31. pii: 636. [Epub ahead of print]16(9):

A Derivatization-Free GC-MS Method for Reliable Quantification of Short-Chain Fatty Acids in Sparus aurata.

Violeta Kalemi, Luca Chiodaroli, Simona Rimoldi, Genciana Terova.

  Background/Objectives: This study presents a gas chromatography-mass spectrometry (GC-MS) method for the derivatization-free quantification of the three major short-chain fatty acids (SCFAs)-acetate, propionate, and butyrate-in gilthead sea bream (Sparus aurata) fecal samples. SCFAs are key metabolites involved in intestinal physiology and are widely recognized as indicators of gut microbiota activity and host health across animal species. In aquaculture research, SCFA profiling can provide valuable insight into diet-microbiota interactions and gut functional status. Methods: The method was developed and optimized using gut-content samples collected from 105 gilthead sea bream at the end of two independent feeding trials, yielding 42 pooled samples (18 from FT1 and 24 from FT2). An extraction protocol based on acidified aqueous extraction with water followed by liquid-liquid extraction using methyl tert-butyl ether was developed (ExA) and subsequently optimized (ExB) to improve extraction consistency and efficiency. SCFA separation and quantification were performed by GC-MS using external calibration. Method validation included evaluation of linearity, matrix effects, and partition coefficients between water and methyl tert-butyl ether. Results: The optimized extraction method (ExB) demonstrated high analytical performance, with linearity coefficients exceeding 0.992 and matrix effects below 17.4%. Conclusions: The proposed method represents a practical and reliable approach for routine SCFA analysis in fish fecal samples. It provides a useful analytical tool for studies investigating diet-microbiota interactions, gut health, and functional responses to nutritional interventions in aquaculture species.

Keywords:  GC-MS chromatography; acetate; butyrate; extraction; fish; gilthead sea bream; propionate; short-chain fatty acid

DOI:  https://doi.org/10.3390/metabo16090636
J Am Soc Mass Spectrom. 2026 Aug 25.

Optimized Sodium-Enhanced MALDI-2 Enables Multimodal Imaging of Triacylglycerols.

Kameron R Molloy, Cody R Marshall, Martin Dufresne, Lukasz G Migas, Matthew S Schrag, Raf Van de Plas, Jeffrey M Spraggins.

  Triacylglycerols (TAGs) are essential neutral lipids with critical roles in cellular function and disease. However, spatial characterization of these chemical species remains difficult because neutral molecules often exhibit low ionization efficiency with common mass spectrometry-based imaging strategies, such as matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Here, we assess how ammonium formate (AmF) or sodium carbonate buffer solution (CBS) tissue washes, combined with varying primary laser power, affect TAG sensitivity with and without MALDI-2 postionization. We found that Na+ doping using Na+ CBS washes combined with MALDI-2 at 31% total laser power was optimal for both signal intensity and molecular coverage. Additionally, Na+ CBS-washed tissues enabled MALDI-2 to be performed using lower laser powers, making this method compatible with sequential microscopy experiments as part of multimodal workflows. Applying this strategy to diseased brain tissue enabled spatial mapping of TAGs linked to Alzheimer's disease. This approach balances sensitivity and tissue preservation, providing a robust platform for multimodal spatial lipidomic studies across diverse tissues and disease contexts.

Keywords:  Alzheimer's disease; MALDI-2; TAGs; cerebral amyloid angiopathy, spatial lipidomics; multimodal imaging; neutral lipids; sensitivity; tissue mapping

DOI:  https://doi.org/10.1021/jasms.6c00227
Drug Test Anal. 2026 Sep 20.

Supercritical Fluid Chromatography for Expedited Sample Clean-Up in Isotope Ratio Mass Spectrometry Including Cholesterol as New Endogenous Reference Compound.

Thomas Piper, Jana Drewes, Mario Thevis.

  Doping control samples showing elevated urinary concentrations and concentration ratios of testosterone or testosterone metabolites are forwarded to isotope ratio mass spectrometry-based determinations in order to differentiate between naturally elevated concentrations and doping offenses. Routine sample preparation protocols commonly encompass liquid-liquid and solid-phase extraction steps and enzymatic deconjugation of steroid glucuronides. In order to separate all steroids of interest from matrix components and to obtain sufficiently clean urinary extracts, high performance liquid chromatography (HPLC) with fraction collection is a frequently employed method of choice. This preparation step is time consuming as each HPLC run requires approx. 45 min, and the evaporation of collected fractions containing water and acetonitrile necessitates up to 60 min. Supercritical fluid chromatography employs supercritical carbon dioxide as eluent combined with other organic solvents as modifiers. The unique physiochemical properties of supercritical carbon dioxide enable the acceleration of the chromatographic separation of different compounds, and collected fractions only contain the modifier and additional methanol as make-up solvent, which allows for rapid evaporation within 10 min. Therefore, a method was developed and validated in-line with current World Anti-Doping Agency-stipulated regulations encompassing testosterone, epitestosterone (E), dehydroepiandrosterone, androsterone, etiocholanolone, 5α- and 5β-androstanediol as target analytes and cholesterol, pregnanediol, 16-androstenol, and 11-oxo-etiocholanolone as endogenous reference compounds. Reference population data encompassed 40 female and 40 male routine doping control samples. Cholesterol as a new endogenous reference compound was carefully investigated. As proof-of-concept, samples derived from a testosterone-gel administration trial were investigated, demonstrating an impact of the drug intervention on E.

Keywords:  cholesterol; doping controls; fraction collection; isotope ratio mass spectrometry; supercritical fluid chromatography

DOI:  https://doi.org/10.1002/dta.70150