bims-metlip 2026-07-26 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–07–26
thirty-one papers selected by
Sofia Costa, Matterworks

Development and Validation of an LC-MS/MS Method for the Quantification of HY-078020 in Human Plasma Using Liquid-Liquid Extraction and Its Application to Human Pharmacokinetic Study.
Automated magnetic-bead-assisted extraction followed by chemical isotope derivatization with 4-(aminomethyl)-N,N-dimethylaniline-d0/d6 for determination of oxylipins derived from n-6 and n-3 polyunsaturated fatty acids by LC-MS/MS.
Direct background subtraction LC-MS/MS assay for human plasma progesterone: Full validation and comparative application.
Development and Validation of a HPLC-MS/MS Method for Quantification of Andrographolide in Rat Plasma by Using Lithium Adduct Detection.
The evolution of DESI-MSI: A comprehensive look at preprocessing and quantification strategies for metabolomics.
Enhanced quantification of 64 pesticides in cannabis-derived oil by isotope-dilution LC-MRM-MS at regulatory decision thresholds.
An ID-HPLC-MS/MS based candidate reference measurement procedure for the quantification of ceramides in human serum.
Comprehensive Liquid Chromatography-Mass Spectrometry-based Metabolite Annotation Elucidates the Chemical Diversity of an Endangered Plant Nardostachys jatamansi.
Challenges in the HPLC analysis of hydrolytically unstable compounds: Metamizole and 4-methylaminoantipyrin case study.
Application of 2D-Chromatography System for the Determination of Cosmetic Peptides in Skin Homogenates.
Efficient dual-mode liquid chromatography-tandem mass spectrometry methods for quantifying major cyanotoxins in natural water.
Towards spatial lipid profiling by using mass spectrometry: analytical challenges and applications.
Mass spectrometry-based metabolomics for studying gut microbiota-derived metabolites in biological samples.
High-throughput mass spectrometry for screening microbial culture supernatants for aromatic amino acids and selected derivatives.
Recent advances in the bioanalysis of acylcarnitines: Methodologies, challenges, and clinical perspectives.
Simple and efficient HPLC-MS/MS method for simultaneous extraction and quantification of anthelmintics in equine manure.
Development and validation of an LC-MS/MS method for the quantification of the KRASG12C inhibitor divarasib.
Development of a liquid chromatography-ion mobility-mass spectrometry method for the screening of d-amino acid alterations in plasma.
A Validated LC-MS/MS Method for Quantifying Phenolic Acids, Lignans, and Enterolignans from Human Fecal Samples.
High-Resolution LC-MS/MS Characterization of Budmunchiamine-Type Alkaloids in Albizia niopoides.
Metabolomics in precision medicine: Application and prospect in cancer research.
Lumefantrine quantification on pre-treated filter paper dried blood spots for clinical field trials.
A systematic DoE approach for optimizing urinary LC-HRMS metabolomics: enhancing reliability in bladder cancer profiling.
Isocitrate Dehydrogenase Mutation Assessment in Human Glioma Using Desorption Electrospray Ionization-Multiple Reaction Monitoring.
Applications of nuclear magnetic resonance spectroscopy in pediatric clinical metabolomics: From research to future perspectives.
Bioanalytical Method for Simultaneous Quantification of Itraconazole and Atorvastatin in Human Plasma.
Liquid Chromatography-Tandem Mass Spectrometry Quantification of Diet-Derived CYP2D6 Biomarkers for Clinical Phenotyping in Japanese Subjects.
Advanced technologies of single-cell metabolomics unveiling cellular metabolic heterogeneity for biological and biomedical research.
Study on the Forced Degradation Behaviour of Oteseconazole and Characterization of Its Degradants by LC-MS/MS.
Broad-spectrum analytical method for the simultaneous determination of multiple classes of urinary volatile organic compound metabolites.
CSU-EP: Contrastive Learning for Unifying Experimental and Predicted EI-MS Spectra.

Biomed Chromatogr. 2026 Sep;40(9): e70549

Development and Validation of an LC-MS/MS Method for the Quantification of HY-078020 in Human Plasma Using Liquid-Liquid Extraction and Its Application to Human Pharmacokinetic Study.

Yaqi Wang, Meiling Zhang, Yazhu Cui, Li Shao, Linying Meng, Mengqin Chen, Mengjie An, Zhijie Shen, Zhaoxing Chu.

  A sensitive and practical liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of HY-078020 in human plasma. Plasma samples (100 μL) were processed by liquid-liquid extraction with methyl tert-butyl ether using HY-078020-d6 as the internal standard. Chromatographic separation was achieved on a Phenomenex Luna C8(2) column with gradient elution, and detection was performed in positive electrospray ionization mode using multiple reaction monitoring. The monitored transitions were m/z 515.2 → 282.3 for HY-078020 and m/z 521.1 → 282.3 for the internal standard. The assay was validated over the concentration range of 4.0-2400 ng/mL with a lower limit of quantification of 4.0 ng/mL. The method showed acceptable selectivity, accuracy, precision, matrix effect, extraction recovery, carryover, and stability in accordance with current bioanalytical validation criteria. Intra- and interbatch precision was within 9.03% and 5.46%, respectively, and the analyte remained stable under benchtop, freeze-thaw, processed-sample, reinjection, and long-term storage conditions. With its small plasma volume requirement, broad calibration range, low carryover, and robust stability, this method is suitable for routine quantification of HY-078020 in human plasma and may support future pharmacokinetic studies.

Keywords:  HY‐078020; LC–MS/MS; bioanalytical method validation; carryover; human plasma; liquid–liquid extraction; stability

DOI:  https://doi.org/10.1002/bmc.70549
J Pharm Biomed Anal. 2026 Jul 16. pii: S0731-7085(26)00327-4. [Epub ahead of print]281 117659

Automated magnetic-bead-assisted extraction followed by chemical isotope derivatization with 4-(aminomethyl)-N,N-dimethylaniline-d0/d6 for determination of oxylipins derived from n-6 and n-3 polyunsaturated fatty acids by LC-MS/MS.

Junhe Chen, Peng Cui, Chengtao Nie, Fan Wu, Qian Wang, Hongwei Zhou, Fangbo Xia.

  Oxylipins derived from n-6 and n-3 polyunsaturated fatty acids (PUFAs) are crucial signaling molecules involved in various physiological and pathological processes. However, the quantification of these metabolites remains challenging due to their low abundance, high structural similarity, and poor electrospray ionization efficiency. Furthermore, existing methods often suffer from complex sample preparation and the lack of cost-effective, reliable internal standards. Therefore, establishing a highly sensitive, automated, and standardized analytical platform for comprehensive oxylipin profiling is the prerequisite for elucidating their complex roles in disease pathogenesis. To address these challenges, we developed a highly sensitive and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, incorporating automated sample preparation through magnetic solid-phase extraction and chemical isotope derivatization using 4- (aminomethyl)-N,N-dimethylaniline-d0/d6 (4-AND-d0/d6) reagents. This approach enables the quantification of 69 oxylipins derived from n-6 and n-3 PUFAs, achieving a remarkable 55- to 1000- fold increase in MS detection sensitivity compared to non-derivatized analysis. Additionally, a retention index (RI)-based predictive model was established to facilitate the screening and identification of unknown regio-isomers. As a proof of concept, this method was applied to quantify oxylipins in serum from a neonatal hypoxic-ischemic encephalopathy (HIE) rat model and to evaluate its applicability in complex biological matrices. Our method offers a sensitive, automated, and reliable tool for oxylipin profiling, with potential applications in biomarker discovery and lipid mediator research. This work presents the first integrated LC-MS/MS strategy combining automated magnetic-bead-assisted extraction and chemical isotopic derivatization for oxylipin analysis, enabling the highly sensitive quantification of 69 target analytes. This study is significant for establishing a robust retention index-based predictive model that facilitates the screening and identification of unknown regio-isomers, offering a reliable and high-throughput analytical solution for clinical biomarker discovery and lipid mediator profiling.

Keywords:  Chemical Isotope Derivatization; HIE Model; LC-MS/MS; Oxylipins; Quantification; Retention Index

DOI:  https://doi.org/10.1016/j.jpba.2026.117659
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Jul 21. pii: S1570-0232(26)00310-7. [Epub ahead of print]1282 125221

Direct background subtraction LC-MS/MS assay for human plasma progesterone: Full validation and comparative application.

Yichao Xu, Xinhua Hu, Pengfei Zhao, Liyuan Zhu, Dandan Yang, Jun Huang, Bo Jiang, Zourong Ruan, Honggang Lou.

   OBJECTIVE: To develop and validate a liquid chromatography-tandem mass spectrometry method based on direct background subtraction for the quantification of endogenous progesterone in human plasma.
METHODS: Protein precipitation was used for sample preparation with deuterated progesterone as the internal standard. Chromatographic separation was performed on an ACQUITY C18 column using gradient elution with 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min. Mass spectrometry was operated in positive electrospray ionization mode with multiple reaction monitoring. Instead of using analyte-stripped matrix or surrogate matrix, authentic plasma was directly used for all validation experiments. Quantitation was achieved by subtracting the background signal, and results were compared with those from the classical method using stripped matrix.
RESULTS: Excellent linearity was achieved over 0.1-100 ng/mL (R2 ≥ 0.99). Precision, accuracy, recovery, matrix effect, and stability all met FDA and ICH M10 acceptance criteria. Compared with the classical method, the bias in Cmax and AUC0-t was within ±15%, indicating no significant difference between the two methods.
CONCLUSION: The direct background subtraction method avoids laborious preparation of blank matrix, eliminates matrix effect discrepancies, and is simple, efficient, and low-cost. It can serve as a general strategy for endogenous substance determination.

Keywords:  Bioanalysis; Human plasma; LC-MS/MS; Method validation; Progesterone

DOI:  https://doi.org/10.1016/j.jchromb.2026.125221
Biomed Chromatogr. 2026 Sep;40(9): e70569

Development and Validation of a HPLC-MS/MS Method for Quantification of Andrographolide in Rat Plasma by Using Lithium Adduct Detection.

Lei Mingdao, Huang Haijin, Luo Xiaoliang, Peng Guoshuang, Sun Yongbing.

  In this study, a simple high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for quantification of andrographolide in rat plasma using andrographolide-lithium adduct as the precursor ion for the first time. The addition of lithium carbonate into the mobile phase could increase the mass response of andrographolide by approximately 100-fold. The analytes were retained and separated on a C18 column and analyzed using a triple quadrupole mass spectrometer equipped with an ESI source. Andrographolide exhibited excellent retention on the C18 column under optimized chromatographic conditions. A simple protein precipitation pretreatment resulted in high extraction recovery (approximately 100%) and negligible matrix effects. The method was linear over the concentration range of 2.00-1606.00 ng/mL. Both precision and accuracy were within acceptable limits. This simple HPLC-MS/MS method has been validated and successfully applied to support pharmacokinetic studies of andrographolide in Sprague-Dawley rats. The present study also provided a strategy for improving the mass response of drugs with low ionization efficiency, especially compounds from plants.

Keywords:  HPLC–MS/MS; andrographolide; lithium adduct ion; pharmacokinetics; protein precipitation

DOI:  https://doi.org/10.1002/bmc.70569
J Food Drug Anal. 2026 Jun 01. 34(1): 55-67

The evolution of DESI-MSI: A comprehensive look at preprocessing and quantification strategies for metabolomics.

Cheng-Hung Yang, Hung-Yao Ho, Chen-Yu Chang, Mei-Ling Cheng.

Mass spectrometry imaging (MSI) has become an indispensable tool in metabolomics for visualizing the spatial distribution of biomolecules within tissues. Due to its direct, ambient analysis capability, desorption electrospray ionization (DESI-MSI) has evolved from a rapid screening tool into a precise and reliable quantitative platform. We delve into its technical principles and particularly review the criticality of sample preprocessing, highlighting the paradigm shift from a "no-preprocessing" approach to one of "optimized preprocessing." The paper details how factors like sample preservation, choice of embedding agents, washing, drying, and on-tissue chemical derivatization all impact data quality. We also discuss how the addition of standards and internal standards can mitigate matrix effects and signal variability, enabling accurate quantitative analysis. Finally, this article looks ahead to the future of DESI-MSI, including its combination with emerging technologies such as tissue expansion mass spectrometry imaging to achieve single-cell-level spatial resolution and open up new possibilities for metabolomics research.

DOI: https://doi.org/10.38212/2224-6614.3583
Anal Bioanal Chem. 2026 Jul 18.

Enhanced quantification of 64 pesticides in cannabis-derived oil by isotope-dilution LC-MRM-MS at regulatory decision thresholds.

Raymond T Suhandynata, Jiaxi Cai, Benjamin J Spiegel, Thomas D Marcotte, Igor Grant, Robert L Fitzgerald, Jeremiah D Momper.

  Cannabis legalization has created regulated markets requiring residual pesticide testing to protect public health. Regulatory programs establish action limits governing product release and enforcement, necessitating accurate quantification in analytically challenging matrices. Compliance is further complicated by the need to quantify dozens of compounds with widely varying physicochemical properties, often requiring multiple assays per product. Validated multiplex methods in representative matrices are therefore critical for defensible quantification at regulatory thresholds. We developed and validated a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 64 pesticides in cannabis-derived hemp oil, employing external calibration with 58 stable isotope-labeled internal standards (SIL-IS) and 4 surrogate internal standards (IS) across 8.4 units of LogP. Validation followed Clinical & Laboratory Standards Institute (CLSI) guidelines (C62-A, EP05-A3, EP29-A) and Food and Drug Administration (FDA) guidance. All 64 pesticides demonstrated linear calibration (R2 ≥ 0.995), with accuracy of 80.0-120.0% (mean recovery = 101.9%) and precision of ≤ 15% coefficient of variation (%CV, mean %CV = 2.7%). Analytical measurement ranges extended to 20,000 parts per billion (ppb) for 62 components. Expanded measurement uncertainty ranged from ±2.3% to ±14.6% and remained ≤15% at 100 ppb for 57 of 64 pesticides. Systematic benchmarking of surrogate IS suitability across all analyte-IS pairings showed that performance correlated with chromatographic and physicochemical similarity, enabling identification of optimal surrogates and analytes requiring matched SIL-IS. This study illustrates an isotope-dilution approach for multiplexed pesticide quantification with structured uncertainty characterization to support accurate and defensible regulatory determinations at regulatory thresholds for cannabis.

Keywords:  Cannabis; Isotope-dilution mass spectrometry; Liquid chromatography-mass spectrometry; Pesticides; Stable-isotope

DOI:  https://doi.org/10.1007/s00216-026-06661-5
Clin Chim Acta. 2026 Jul 22. pii: S0009-8981(26)00425-0. [Epub ahead of print] 121243

An ID-HPLC-MS/MS based candidate reference measurement procedure for the quantification of ceramides in human serum.

Huoyan Ji, Xiaozhen Zhang, Xiaofan Yin, Xiaodong Yang, Weiming He, Min Shen.

   BACKGROUND: Ceramides have emerged as promising biomarkers for predicting residual cardiovascular risk in addition to traditional lipid parameters. We developed and validated a candidate reference measurement procedure (RMP) based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for the accurate quantification of four ceramide subspecies (Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1)) relevant to cardiovascular risk in human serum.
METHODS: Primary reference materials (PRMs) were characterized by quantitative nuclear magnetic resonance (qNMR) to establish SI traceability. A ChromCore AQ C18 column with gradient elution was used. Method validation followed Clinical and Laboratory Standards Institute (CLSI) guidelines, assessing specificity, matrix effects, linearity, limits of quantification (LOQ) and detection (LOD), trueness, precision, and stability.
RESULTS: Excellent linearity was observed across the following ranges: 19.90-4081.48 nmol/L for Cer(d18:1/16:0), 9.92-2022.39 nmol/L for Cer(d18:1/18:0), 49.99-9877.01 nmol/L for Cer(d18:1/24:0), and 49.94-9873.91 nmol/L for Cer(d18:1/24:1), with R2 = 1.00 for all analytes. LOQs were 15.22 nmol/L, 7.63 nmol/L, 37.09 nmol/L, and 38.44 nmol/L, respectively. Recovery rates ranged from 95.04% to 101.15%, with intra-day and inter-day precision (CV) below 3.0% and 4.0%, respectively. Analysis of NIST SRM 1950 yielded Z-scores of 0.9-1.7. Expanded uncertainties ranged from 4.72% to 8.52% (k = 2). Reference intervals (P2.5-P97.5) were 187.10-442.50 nmol/L, 35.30-140.05 nmol/L, 1374.63-4522.55 nmol/L, and 585.30-1906.66 nmol/L, respectively.
CONCLUSION: The candidate RMP was successfully developed and validated for the accurate quantification of four ceramide subspecies relevant to cardiovascular risk in human serum, with SI traceability.

Keywords:  Candidate reference measurement procedure; Cardiovascular risk; Ceramides; Isotope dilution liquid chromatography-tandem mass spectrometry; Metrological traceability

DOI:  https://doi.org/10.1016/j.cca.2026.121243
J Sep Sci. 2026 Jul;49(7): e70494

Comprehensive Liquid Chromatography-Mass Spectrometry-based Metabolite Annotation Elucidates the Chemical Diversity of an Endangered Plant Nardostachys jatamansi.

Yulin Cui, Kouharu Otsuki, Mi Zhang, Chang Luo, Siyi Li, Takashi Kikuchi, Jie Zhang, Wei Li.

  Nardostachys jatamansi is an endangered aromatic plant that produces a chemically complex and diverse array of secondary metabolites. In this study, we aimed to comprehensively characterize the chemical diversity of N. jatamansi by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolite annotation. A metabolite annotation workflow combining feature detection, library matching, MetFrag-based in silico fragmentation, SIRIUS fragmentation tree analysis, manual MS/MS interpretation, molecular networking, chemical classification, and structural validation of selected compounds was applied to methanolic extracts of N. jatamansi. Using this integrated strategy, 111 peaks were structurally annotated, and an additional 13 peaks were assigned to tentative structural level annotations based on MS/MS fragment similarity in a molecular network, giving a total of 124 peaks classified at the chemical structural level. The annotated metabolites mainly comprised iridoids, lignans, sesquiterpenoids, sesquiterpenoid hybrids, monoterpenoids, flavonoids, and other constituents such as fatty acids and phenylpropanoids, revealing the remarkable chemical diversity of N. jatamansi. The annotation performance of each metabolite annotation platform was systematically compared in terms of coverage, efficiency, and chemical class level specificity, revealing that these platforms provided complementary annotations whose combined use with molecular networking greatly enhanced metabolite coverage and structural interpretation. This integrated LC-MS/MS and multi-platform workflow extends current knowledge of the chemical diversity of N. jatamansi.

Keywords:  LC‐MS/MS; MetFrag; Nardostachys jatamansi; SIRIUS; molecular networking

DOI:  https://doi.org/10.1002/jssc.70494
J Pharm Biomed Anal. 2026 Jul 14. pii: S0731-7085(26)00315-8. [Epub ahead of print]281 117647

Challenges in the HPLC analysis of hydrolytically unstable compounds: Metamizole and 4-methylaminoantipyrin case study.

Michal Douša, Jakub Jireš.

  The on-column hydrolytic degradation of metamizole to impurity C in aqueous mobile phases represents a major source of analytical errors and false-positive results. Accurate and precise determination of impurity C is critical because this analyte is a direct precursor to a monitored genotoxic nitrosamine impurity. To resolve this issue, a novel isocratic non-aqueous hydrophilic interaction liquid chromatography method was developed on a triazole-bonded stationary phase. The separation was performed on Cosmosil HILIC column column (150 × 3.0 mm, 2.5 µm). The mobile phase was composed of 10 mM ammonium formate and 5 mM formic acid in a mixture of methanol and acetonitrile (3:97, v/v). An isocratic flow rate of 0.8 mL/min was utilized at a column temperature of 10 °C. A short analysis time of 3 min was achieved under optimized conditions. Impurity C exhibited minimal retention (k = 0.4), whereas metamizole was strongly retained on the column (k > 100). Due to this distinct selectivity, on-column artifact formation was completely prevented throughout the chromatographic run. To manage metamizole accumulation on the stationary phase, a periodic column regeneration step was introduced after every 30 sample injections. The method was coupled with mass spectrometric detection in positive electrospray ionization mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 218 as [M+H]+. A low limit of quantification of 2 ng/mL (2 ppm) and excellent linearity (r = 0.9991) were obtained. The calculated back-fit bias remained between -2.6% and 2.3% across the entire calibrated range. Critical verification parameters demonstrated full compliance with International Council for Harmonisation guidelines. The practical utility of the procedure was confirmed through the analysis of two production batches of metamizole.

Keywords:  Dynamic chromatogram; Impurity C (4-methylaminoantipyrin); Metamizole; NA-HILIC; Separation of unstable compounds

DOI:  https://doi.org/10.1016/j.jpba.2026.117647
J Mass Spectrom. 2026 Aug;61(8): e70094

Application of 2D-Chromatography System for the Determination of Cosmetic Peptides in Skin Homogenates.

Fosca Errante, Marco Pallecchi, Marta Menicatti, Lisa Giovannelli, Paolo Rovero, Gianluca Bartolucci.

  Peptides are increasingly employed as active ingredients in both therapeutic and cosmeceutical applications due to their high biological specificity, favorable safety profiles, and expanding market relevance. In topical formulations, peptide activity is typically confined to the skin, where widespread dermal proteases may significantly affect their stability and efficacy. Despite the growing use of bioactive peptides in dermatological and cosmeceutical products, robust analytical methodologies for assessing their susceptibility to dermal enzymatic degradation remain limited. In this study, a two-dimensional HPLC-MS/MS (2D-HPLC-MS/MS) method based on ion trap detection was developed and validated for the quantitative evaluation of peptide stability in human skin homogenate (HSH). The analytical setup integrates online cleanup with chromatographic separation on a silica-based pentafluorophenyl (PFP) column, enabling reliable analysis of peptides with different polarity profiles within a single workflow. The system enables direct injection of sample solutions with relatively high organic solvent content, allowing limited sample dilution and preservation of analytical sensitivity. A key feature of the method is the use of isomeric peptide analogues as internal standards, monitored under identical MS/MS conditions as their corresponding analytes. This approach provides effective correction for ionization and fragmentation variability while offering a practical alternative to stable isotope-labelled standards. The ion trap mass analyzer ensured controlled and reproducible fragmentation behavior, supporting robust quantitative performance. The developed method demonstrated satisfactory linearity, sensitivity, precision, accuracy, and minimal matrix effects. Application to stability studies of three peptides with distinct polarity profiles confirmed its suitability for monitoring degradation kinetics in HSH. Overall, this 2D-HPLC-MS/MS strategy provides a versatile analytical platform for dermal peptide stability assessment and supports early-stage screening and preclinical studies of bioactive peptides intended for topical applications.

Keywords:  dermal stability; ion trap mass spectrometry; isomeric peptide analogues; online sample cleanup; scrambled peptide

DOI:  https://doi.org/10.1002/jms.70094
J Chromatogr A. 2026 Jul 08. pii: S0021-9673(26)00574-1. [Epub ahead of print]1785 467245

Efficient dual-mode liquid chromatography-tandem mass spectrometry methods for quantifying major cyanotoxins in natural water.

Dani Khoury, Christelle Pallez, Sophie Lardy-Fontan, Christophe Rosin.

  Cyanobacterial blooms in freshwater resources frequently originate from toxin-producing strains capable of generating chemically diverse cyanotoxins. These blooms not only disrupt aquatic ecosystems but also pose significant risks to public health due to the production of hepatotoxins (such as microcystins (MCs) and nodularins (NODs)) and neurotoxins (such as anatoxins (ATXs), saxitoxins (STXs), and cylindrospermopsins (CYNs)). It is therefore essential to accurately detect and quantify cyanotoxins in order to assess exposure risks, inform water management decisions, and guide public health interventions. However, this task remains analytically challenging due to the wide polarity range of these compounds (-5.1 < XLogP < 5.2), their structural diversity, and their presence at trace levels (ng L⁻1). In this study, we developed and validated two high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods for the quantification of 24 priority cyanotoxins across diverse families. The panel included eleven MC variants, one NOD, three ATXs, three CYNs, and six STX analogs. Peptides (MCs and the NOD) were analyzed using reversed-phase online solid-phase extraction (SPE)-HPLC-MS/MS, whereas more polar toxins (ATXs, STXs, and CYNs) were analyzed using direct injection in hydrophilic interaction liquid chromatography mode (DI-HPLC-MS/MS) after minimal sample preparation. Quantification was performed using isotope-labeled surrogate internal standards, with one representative labeled analogue assigned to each toxin class to improve correction for matrix effects and analytical variability. Both methods were optimized and validated following SANTE/EURL guidelines, using representative water matrices. Calibration curves exhibited close quadratic fits (R² > 0.99). Mean recoveries ranged from 77 to 120% with relative standard deviations below 15% and maximum measurement uncertainties under 40% for most analytes. Quantification limits reached 20 ng L⁻1 for MCs and the NOD, and ranged from 50 to 250 ng L⁻1 for ATXs, CYNs, and STXs. Application of these methods to the summer monitoring campaign for 2025 in the Grand-Est region of northeastern France confirmed their robustness and successfully quantified six MC variants, two STX analogs, and ATX in bathing waters.

Keywords:  Drinking water; Hydrophilic-interaction liquid chromatography; Method validation; Multiclass cyanotoxins; Online solid-phase extraction; Surface water

DOI:  https://doi.org/10.1016/j.chroma.2026.467245
Anal Bioanal Chem. 2026 Jul 23.

Towards spatial lipid profiling by using mass spectrometry: analytical challenges and applications.

Yaowei Liu, Yihua Ou, Hemi Luan, Tiangang Luan.

  Lipidomics, as a crucial branch of metabolomics, is dedicated to systematically analyzing the composition, structure, function, and dynamic changes of lipids in organisms, playing a pivotal role in elucidating disease mechanisms and discovering biomarkers. Conventional lipidomics methods based on liquid chromatography-mass spectrometry (LC-MS) require tissue homogenization, which obscures the spatial distribution of lipids and precludes the analysis of their heterogeneity within complex tissue microenvironments. In recent years, the development of spatial omics technologies such as mass spectrometry imaging (MSI) and laser capture microdissection (LCM) has provided powerful tools for the in situ and visual investigation of lipid spatial distribution. This paper systematically reviews the main analytical strategies in lipidomics, focusing on the technical principles, advances, and recent applications of spatial multi-omics integration. It further discusses the challenges faced by spatial lipidomics in terms of quantitative accuracy, isomer identification, and spatial localization precision, and provides an outlook on future technological developments. Spatial lipidomics breaks through the bottleneck of losing spatial information in traditional methods, and opens up a new path for further exploration of disease mechanisms and the discovery of new biomarkers in the spatial dimension.

Keywords:  Laser capture microdissection; Lipid metabolism; Mass spectrometry imaging; Spatial lipidomics; Spatial multi-omics

DOI:  https://doi.org/10.1007/s00216-026-06684-y
J Food Drug Anal. 2026 Jun 01. 34(1): 2-19

Mass spectrometry-based metabolomics for studying gut microbiota-derived metabolites in biological samples.

Ya-Ting Lin, Huai-Hsuan Chiu, Chih-Ning Cheng, Ching-Hua Kuo.

Gut microbiota produces a wide range of metabolites and plays a critical role in maintaining host health. Dysregulation of these metabolites can influence host metabolism through systemic circulation, contributing to the development of various diseases, including immunological, neurological and cancer-related disorders. Chromatography coupled with mass spectrometry (MS) has emerged as a powerful analytical approach, offering high sensitivity and resolution for studying gut microbiota-related metabolites. This review provides a comprehensive overview of chromatographic MS-based methods applied to the study of the gut microbial metabolome. We summarize strategies for sample collection, storage, and preparation of commonly analyzed sample types, including feces, plasma/serum, urine, and tissue samples. In addition, we included the main chromatographic MS-based approaches, as well as data analysis techniques, for investigating the gut microbial metabolome. The characteristics and utility of liquid chromatographicmass spectrometry (LC-MS), gas chromatographic-mass spectrometry (GC-MS), and capillary electrophoresis-mass spectrometry (CE-MS) were discussed in the context of providing broader coverage of gut microbiota-derived metabolites with diverse physicochemical properties. Finally, we summarize recent studies that have employed chromatographic MS-based approaches to investigate gut microbiota-related disease. Through the integration of appropriate sample handling and advanced analytical strategies, a deeper understanding of host-microbiota interactions and their roles in health and disease can be achieved.

DOI: https://doi.org/10.38212/2224-6614.3568
Biotechnol Rep (Amst). 2026 Sep;51 e00969

High-throughput mass spectrometry for screening microbial culture supernatants for aromatic amino acids and selected derivatives.

Amisha Patel, Stefan Stagge, Payam Ghiaci, Leif J Jönsson.

  Sustainable production of aromatic amino acids (AAAs) and their derivatives are central to developing renewable routes for aroma- and flavour-related chemicals with myriads of applications in pharmaceutical industry, food and nutrition, cosmetics and personal care, polymer synthesis, and biotechnology. The aim of the present study was to establish and validate a high-throughput mass spectrometry (HT-MS) workflow for rapid screening of AAAs and selected aromatic derivatives, i.e., kynurenic acid, indole-3-acetic acid, and p-coumaric acid, using an automated solid-phase extraction system coupled to time-of-flight detection. A method based on fast cycle time, 15 s per sample, and minimal sample handling was elaborated to enable high-throughput screening of large culture collections. Validation using pure standards confirmed excellent linearity (R² ≥ 0.99), accuracy, precision, and on-instrument stability at 4 °C. Although strong matrix effects were observed in microbial culture supernatants, analyte detection remained selective and reproducible, supporting reliable semi-quantitative screening. The results demonstrate that the proposed workflow supports semi-quantitative, comparative analysis of AAAs and their derivatives across large sample sets. Overall, the developed HT-MS method provides a robust and efficient platform for rapid pre-screening of microbial collections accelerating the discovery and optimization of aromatic metabolite production.

Keywords:  Aromatic amino acids; High-throughput; Indole-3-acetic acid; Kynurenic acid; Mass spectrometry; Microbial screening; p-Coumaric acid

DOI:  https://doi.org/10.1016/j.btre.2026.e00969
Pharm Sci Adv. 2026 Dec;4 100130

Recent advances in the bioanalysis of acylcarnitines: Methodologies, challenges, and clinical perspectives.

Jiaxin Zheng, Wanting Chen, Fengxia Qin, Pei Zhang, Fengguo Xu.

  Acylcarnitines (ACs) are a diverse class of fatty acid esters of L-carnitine that serve as critical mediators in energy homeostasis and mitochondrial function. Beyond their classical role in newborn screening for inborn errors of metabolism, ACs have emerged as promising biomarkers for complex pathologies, including cardiovascular diseases, diabetes, and drug-induced toxicities. However, the accurate quantification and comprehensive profiling of ACs in biological matrices remain analytically challenging due to their broad polarity range, vast concentration disparities, and the presence of isomers. This review provides a comprehensive overview of the current bioanalytical strategies for ACs, covering sample preparation techniques and detection platforms, with a focus on liquid chromatography-mass spectrometry. Special attention is given to the differentiation of isomers. Finally, we discuss the clinical applications of ACs profiling and highlight future perspectives, including the integration of ion mobility spectrometry, automated high-throughput workflows, spatial and single-cell metabolomics, and AI-driven analytics, to pave the way for precision medicine.

Keywords:  Acylcarnitines; Clinical diagnosis; Mass spectrometry; Metabolic disease; Sample pretreatment

DOI:  https://doi.org/10.1016/j.pscia.2026.100130
MethodsX. 2026 Dec;17 104042

Simple and efficient HPLC-MS/MS method for simultaneous extraction and quantification of anthelmintics in equine manure.

K Holzer, U Bertsche, M Meyer, T Schilling, J Pfannstiel, L E Hoelzle.

  Anthelmintic treatment is a common practice in horses. The presence of pharmaceutical residues in fields and pastures is well-documented affecting various organisms, like nematodes. This study describes the development and validation of a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method using multiple reaction monitoring (MRM) for extracting and determining the concentrations of four commonly used anthelmintics in horse manure. Manure was initially dried, then spiked with various concentrations (10-10,000 ng) of anthelmintics and extracted using an acetonitrile-water mixture containing 5 mM ammonium formate, with no need for further solid-phase extraction (SPE) cleaning. Recovery rates ranged from 82.3% for pyrantel to 120.8% for ivermectin, with standard deviations below 20%. Calibration curves were linear between 1 ng/mL and approximately 1.5 µg/mL for all four components. The lower limits of detection (LLODs) were 0.1 ng/mL for ivermectin, 0.2 ng/mL for moxidectin, 1.0 ng/mL for fenbendazole due to carryover, and 0.3 ng/mL for pyrantel, which formed two peaks.•Novel method for simultaneous extraction of anthelmintics from horse manure.•Method allows efficient and reliable detection of the mainly used anthelmintics in horses.•Basis for the extension of the method to other applications, including the analysis of anthelmintics in soil samples.

Keywords:  Anthelmintic; Extraction; Fenbendazole; HPLC-MS/MS; Horse manure; Ivermectin; MRM; Moxidectin; Pyrantel pamoate

DOI:  https://doi.org/10.1016/j.mex.2026.104042
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Jul 18. pii: S1570-0232(26)00300-4. [Epub ahead of print]1282 125211

Development and validation of an LC-MS/MS method for the quantification of the KRASG12C inhibitor divarasib.

Jamie Rijmers, Viët Bui, Maria C Lebre, Alfred H Schinkel, Jos H Beijnen, Olaf van Tellingen, L J van Winden.

  Divarasib is a newly developed covalent KRASG12C inhibitor, currently under clinical investigation in a phase 3 trial in patients with non-small cell lung cancer (NSCLC). At the moment, very limited pharmacokinetic data are publicly known. However, obtaining more insight into the pharmacokinetic properties of divarasib is important, since this may provide a better understanding of its efficacy and safety risks. Pre-clinical studies have been performed in mouse models to evaluate the effect of drug transporters and drug-metabolizing enzymes on the plasma exposure and tissue distribution of divarasib. Therefore, a reliable quantification method is required. To our knowledge, no bioanalytical assay of divarasib has been published yet. Therefore, in this study we developed and validated an assay to quantify divarasib in human plasma and in eight different mouse-related matrices, and partially in mouse plasma, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was initially evaluated over a concentration range of 1-10,000 nM. However, due to carry-over observed at 10,000 nM, the validated calibration range was established at 1-2000 nM, with matrix-dependent LLOQs of 1-10 nM. Erlotinib was used as an internal standard and acetonitrile was utilized to perform protein precipitation as sample pretreatment. Divarasib demonstrated stability in human plasma and in mouse plasma and tissue homogenates under various experimental conditions. A pilot in vivo study showed the applicability of our validated LC-MS/MS method. Ongoing clinical trials may collect plasma samples, and this developed method enables quantification of divarasib in both mouse and human plasma samples.

Keywords:  Bioanalysis; Divarasib; KRAS(G12C); LC–MS/MS; Mouse matrices

DOI:  https://doi.org/10.1016/j.jchromb.2026.125211
J Chromatogr A. 2026 Jul 16. pii: S0021-9673(26)00606-0. [Epub ahead of print]1785 467278

Development of a liquid chromatography-ion mobility-mass spectrometry method for the screening of d-amino acid alterations in plasma.

Luisa-Gabriela Bogos, Ioana-Ecaterina Pralea, Alina-Ramona Potra, Ina Maria Kacso, Radu-Cristian Moldovan, Cristina-Adela Iuga.

  Probing chirality, one of the fundamental properties of all biological systems, remains analytically challenging and often requires specialized analytical setups or reagents, limited to few laboratories. This study aimed to develop an efficient liquid chromatography coupled to ion-mobility mass spectrometry (LC-IM-MS) method for the simultaneous analysis of all proteinogenic d-amino acids (d-AAs) in human plasma, using exclusively commercially available reagents. An indirect enantioseparation strategy was developed based on derivatization with (S)-N-(4-nitrophenoxycarbonyl) phenylalanine methoxyethyl ester ((S)-NIFE), with five stationary phases evaluated for diastereomer separation capability. A design of experiments approach was employed for further optimization, achieving baseline separation of all proteinogenic AA diastereomers within 20 min on a phenyl-based stationary phase. A straightforward sample preparation protocol was characterized, then the entire workflow was used for the assessment of d-AAs alterations across 60 plasma samples obtained from patients with chronic kidney disease (CKD, stages 4 and 5) and healthy controls. The analysis revealed increased levels of d-Pro, d-Arg, d-Ser, d-Asn, d-Gln, and d-Ala in CKD samples, highlighting the effectiveness of the developed analytical workflow to detect disease-related changes in plasmatic d-AAs.

Keywords:  Chiral analysis; Chiral derivatizing reagent; Chronic kidney disease; Indirect enantioseparations; Metabolomics; d-amino acids

DOI:  https://doi.org/10.1016/j.chroma.2026.467278
Acs Nutr Sci. 2026 Jul 21. 1(4): 356-366

A Validated LC-MS/MS Method for Quantifying Phenolic Acids, Lignans, and Enterolignans from Human Fecal Samples.

Christopher A Dicksion, Darrian N Chao, Jonathan D Rickmeyer, Elizabeth N Bess.

  The human gut is home to numerous small molecules that impact health. Three prominent classes of molecules in this environment are phenolic acids, lignans, and enterolignans, which have been linked to anti-inflammatory and antioxidant effects as well as protection from cancer, cardiovascular disease, and neurodegeneration. The abundance of these molecules in the intestine as well as their biological significance motivated the development and validation of the LC-MS/MS method reported herein, which provides a simple, robust, and high-throughput approach to simultaneously quantify a 22-membered panel of phenolic acids, lignans, and enterolignans in human fecal samples. Facile sample preparation and a short analytical time (5 min per sample, compared to similar methods that range from 7.8-28 min) allow for high throughput. A 16-fold increase in sensitivity allows for quantitation of lignans that are often not detected via existing methods.

Keywords:  LC−MS/MS; enterolignans; fecal samples; human gut microbiome; lignans; phenolic acids

DOI:  https://doi.org/10.1021/acsnutrsci.6c00063
Rapid Commun Mass Spectrom. 2026 Oct 15. 40(19): e70142

High-Resolution LC-MS/MS Characterization of Budmunchiamine-Type Alkaloids in Albizia niopoides.

Maria L A Majevski, Lienne D'Auria Lima, Adriana C C Reis, Leonan I C R Santos, Brenno F S Vargas, Markus Kohlhoff, Geraldo Célio Brandão.

   RATIONALE: Budmunchiamines are macrocyclic spermine alkaloids that constitute a distinctive class of polyamine natural products predominantly found in species of the genus Albizia. Comprehensive characterization of these compounds remains challenging because of their structural diversity and the limited availability of reference standards. This study aimed to profile the budmunchiamine alkaloids present in the trunk bark of Albizia niopoides using high-resolution tandem mass spectrometry.
METHODS: Alkaloids were extracted from the trunk bark of A. niopoides and analyzed by ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) operated in positive ionization mode. Compound annotation was based on accurate mass measurements (≤ 5 ppm), chromatographic retention behavior, isotopic patterns, and diagnostic MS/MS fragmentation, supported by comparison with published data.
RESULTS: Twenty-six macrocyclic spermine alkaloids were detected and annotated. The MS/MS spectra consistently exhibited characteristic budmunchiamine fragment ions at m/z 214, 171, 169, and 129, together with diagnostic neutral losses, confirming assignment to this structural class. Six compounds were identified as the known budmunchiamines L1, L4, L5, and L6, whereas two corresponded to previously synthesized analogues (Compounds 2 and 21). The remaining 20 compounds displayed distinct precursor ions but closely related fragmentation pathways, indicating previously unreported budmunchiamine-type analogues.
CONCLUSIONS: UHPLC-ESI-QTOF-MS/MS proved to be an effective strategy for the dereplication and structural annotation of macrocyclic spermine alkaloids from A. niopoides. The results substantially expand the known chemical diversity of budmunchiamines in this species and demonstrate the value of high-resolution tandem mass spectrometry for the characterization of structurally complex natural products.

Keywords:   Albizia niopoides ; budmunchiamines; high‐resolution mass spectrometry; macrocyclic spermine alkaloids; natural product dereplication

DOI:  https://doi.org/10.1002/rcm.70142
J Food Drug Anal. 2026 Jun 01. 34(1): 20-30

Metabolomics in precision medicine: Application and prospect in cancer research.

Ming-Shi Shiao.

Metabolomics is the most recent and final discipline among the post-genomic, multiomics fields. It encompasses key technologies in systems biology, providing an overview of low-molecular-weight metabolites while examining disturbances to offer pathophysiological explanations. The two key analytical tools in metabolomics are nuclear magnetic resonance spectroscopy and mass spectrometry, which includes gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Metabolomic research relies on a three-stage data generation process: the first stage involves precise analytical chemistry to produce data; the second stage employs multivariate statistical analysis; and the last stage uses big data as a reference for comparison to establish biochemical, metabolic, and pathophysiological correlations, thereby providing possible interpretations. The initiative of precision medicine starts with cancer as the first disease target. This review discusses and illustrates the application of metabolomics in precision medicine, choosing two types of cancer as example. Warburg effect and metabolic reprogramming are particularly discussed. By providing global, top-down, and less biased information, metabolomics enables precision medicine.

DOI: https://doi.org/10.38212/2224-6614.3584
Malar J. 2026 Jul 20.

Lumefantrine quantification on pre-treated filter paper dried blood spots for clinical field trials.

Daniel Blessborn, Natpapat Kaewkhao, Joel Tarning.

  Artemether-lumefantrine is the most widely used antimalarial treatment for uncomplicated Plasmodium falciparum malaria. Malaria predominantly affects populations in rural areas of tropical and subtropical regions. Drug resistance is a growing concern, and monitoring antimalarial efficacy remains crucial for detecting changes in treatment performance and tracking potential resistance trends. In this study, we present an efficient method for collecting blood samples in resource-limited settings, using dried blood spots on pre-treated filter paper, which enhances the stability of lumefantrine. This approach builds on previous methods by incorporating rapid sample processing through semi-automated punching robots for 96-well plate formats, quick sample cleanup via filtration through a phospholipid removal plate, and expedited analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a turnaround time of just 2 min per sample. The method also achieves a significantly improved the lower limit of quantification of 15 ng of lumefantrine per ml of blood (i.e. LLOQ of 15 ng/ml). A Phenomenex Luna-CN 50 × 2 mm, 3 µm LC column was used, with a flow rate of 400 µl/min and a mobile phase consisting of acetonitrile and ammonium formate buffer (10 mM, 0.5% formic acid) in a 67:33 (v/v) ratio. The linear range for quantification spanned from 15 to 15,000 ng/ml, based on three 3.2 mm punched discs from a 50 µl blood spot. Accuracy was within ± 15% of the validation criteria, and precision was demonstrated by intra-assay variability of less than 7.2% and inter-assay variability of less than 12% across all concentration levels. The stability of lumefantrine in dried blood spots on pre-treated filter paper was demonstrated using a sample set obtained from another research group, which had been stored in the dark under low-humidity conditions for up to 16 years. The method showed no endogenous interference or interference from the concomitant drug artemether and is suitable for use in clinical trials.

Keywords:  Dried blood spots; Filter paper; Lumefantrine; Malaria

DOI:  https://doi.org/10.1186/s12936-026-06048-y
Anal Bioanal Chem. 2026 Jul 24.

A systematic DoE approach for optimizing urinary LC-HRMS metabolomics: enhancing reliability in bladder cancer profiling.

Anastasiia Frolova, Mikhail Vokuev, Yurii Ikhalainen, Daria Prosuntsova, Igor Rodin.

  Untargeted urinary metabolomics presents significant challenges in analytical reproducibility and biological interpretation, particularly in the context of clinical oncology. This study presents a systematically optimized liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflow for bladder cancer (BCa) biomarker discovery. To address variability in sample preparation, a two-stage design of experiments (DoE) approach was applied to systematically optimize key parameters affecting metabolite extraction efficiency, thereby improving the reproducibility of subsequent non-invasive profiling. The performance of the workflow was evaluated through the systematic assessment of instrumental stability and injection precision using pooled quality control (QC) samples. Following peak picking and alignment, a comprehensive raw dataset of 15,344 metabolic signals was generated, leading to the putative identification of 854 compounds. Unsupervised principal component analysis (PCA) demonstrated reproducible instrumental performance, indicated by tight QC sample clustering. From the total clinical cohort of 107 patients, a demographically matched sub-cohort of 50 individuals was evaluated to suppress confounding physiological noise. This comparative model revealed distinct disease-specific clustering and demonstrated significant perturbations in the tryptophan metabolic axis, membrane lipid remodeling, and enhanced proteolytic activity, characterized by an evident peptide overflow, associated with BCa progression. This systematically optimized methodology provides a reliable analytical approach for identifying non-invasive diagnostic panels, supporting the implementation of efficient laboratory workflows aligned with Analytics 5.0 principles.

Keywords:  Analytics 5.0; Bladder cancer biomarkers; Design of experiments (DoE); LC-HRMS; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s00216-026-06678-w
Anal Chem. 2026 Jul 20.

Isocitrate Dehydrogenase Mutation Assessment in Human Glioma Using Desorption Electrospray Ionization-Multiple Reaction Monitoring.

Mahdiyeh Shahi, Diogo Moniz Garcia, Alfredo Quiñones-Hinojosa, R Graham Cooks.

The mutation status of isocitrate dehydrogenase (IDH) in glioma patients has diagnostic and prognostic significance but is available only after surgery. Rapid and sensitive intraoperative molecular diagnosis is essential for distinguishing tumor from infiltrative and from normal brain tissue to maximize safe resection. IDH-mutant (mut)tumors produce high concentrations of 2-hydroxyglutarate (2HG), which serves as a surrogate marker of IDH mutation. Here, we use desorption electrospray ionization-multiple reaction monitoring (DESI-MRM), a form of mass spectrometry (MS), for rapid prediction of IDH mutation status. A set of 30 human brain smears was analyzed using DESI-MRM experiments to predict the mutation status of IDH and quantify 2HG in glioma samples within 2 min (endogenous reference molecule) or 7 min (external standard). Nanoelectrospray ionization (nESI) was employed as a validation method. An isotopically labeled surrogate internal standard allowed quantitative measurement of 2HG and clear separation between IDH-mut and IDH-wildtype (wt) samples, showing 100% agreement with clinical diagnosis. 2HG concentrations in IDH-mut samples ranged from 88 to 1445 ng mg-1, while IDH-wt samples exhibited 2HG concentrations of 9-15 ng mg-1. Calibration curves showed method accuracy within ≤15%. The quantitative results obtained using isotopically labeled standards showed a strong correlation between IDH mutation score and 2HG concentrations, as did an earlier semiquantitative (ratiometric, endogenous standard) method. These findings highlight the standalone capability of DESI-MRM for making measurements to justify assessments for intraoperative diagnosis and to support surgical decision-making to improve patient outcomes.

DOI: https://doi.org/10.1021/acs.analchem.6c00520
J Food Drug Anal. 2026 Jun 01. 34(1): 68-77

Applications of nuclear magnetic resonance spectroscopy in pediatric clinical metabolomics: From research to future perspectives.

Chieh-Ni Kuo, Meng-Han Chiang, Hsien-Ju Lee, Yu-Ying Yu, Eric Yi-Liang Shen, Chih-Yung Chiu.

Metabolomics studies small-molecule metabolites to provide insights into health and disease, supporting early diagnosis and personalized medicine. Advances in mass spectrometry and nuclear magnetic resonance (NMR) have expanded its use in metabolic, cancer, and cardiovascular diseases. In pediatrics, high-resolution NMR metabolomics has been instrumental in identifying age-related metabolic changes during early childhood and their associations with growth, nutrition, and disease risk. However, a comprehensive review of its clinical applications and future potential remains limited. This review highlights how utilizing specific NMR pulse sequences, such as CPMG and NOESY, allows for precise and non-destructive metabolic profiling of diverse biofluids, supported by minimal sample preparation and high-throughput automated analysis. Data processing tools like NMRProcFlow and MetaboAnalyst facilitate spectral preprocessing, statistical analysis, and biological interpretation, streamlining metabolomics workflows. Clinically, NMR-based metabolomics has elucidated metabolic alterations in pediatric growth, prematurity, nutrition-related sensitizations, allergic diseases, lipid metabolism, infectious conditions, and neurobehavioral disorders. In particular, metabolomics has been applied to identify specific metabolic signatures underlying the molecular mechanisms of childhood allergic asthma. Despite limitations in detecting low-abundance metabolites, NMR's ability to preserve sample integrity and integrate multi-omics data, especially gut microbiota-derived metabolites, shows great promise in advancing precision pediatric medicine, early disease screening, and personalized therapeutic strategies.

DOI: https://doi.org/10.38212/2224-6614.3574
Drug Metab Bioanal. 2026 ;19(1): 79-93

Bioanalytical Method for Simultaneous Quantification of Itraconazole and Atorvastatin in Human Plasma.

Kunjan B Bodiwala, Flora J Pujara, Avinash D Khadela, Shrinivas S Savale.

   INTRODUCTION: Drug-drug interactions between statins and azole antifungal agents, due to the inhibition of the CYP3A4 enzyme by antifungals, increase the risk of adverse effects from statins. Thus, a precise bioanalytical method for quantifying Itraconazole and Atorvastatin in human plasma is essential.
MATERIALS AND METHODS: A liquid chromatographic method with protein precipitation and liquidliquid extraction was developed. Rosuvastatin calcium served as the internal standard. The method was validated per ICH M10 and USFDA guidelines and used to analyze spiked plasma samples.
RESULTS: The method effectively separated Itraconazole, Atorvastatin, and the internal standard without plasma interference. It achieved linear responses for each drug in the 0.1-3.0 μg/mL range with regression coefficients > 0.99. The RSD for within-run and between-run responses was < 5%, and the average recovery exceeded 64%.
DISCUSSION: The method accurately and precisely measured each analyte at the LLOQ level (0.1 μg/mL). A sensitive and selective bioanalytical HPLC method was developed, validated, and applied for the simultaneous estimation of Itraconazole and Atorvastatin in human plasma.
CONCLUSION: This method ensures safe and effective co-administration of these medications in clinical practice, benefiting patient care.

Keywords:  Bioanalytical method; LLE; UFLC; atorvastatin calcium; itraconazole; rosuvastatin calcium

DOI:  https://doi.org/10.2174/0118723128401590250922065135
Clin Transl Sci. 2026 Aug;19(8): e70668

Liquid Chromatography-Tandem Mass Spectrometry Quantification of Diet-Derived CYP2D6 Biomarkers for Clinical Phenotyping in Japanese Subjects.

Masaki Kumondai, Nagomi Hayashi, Yotaro Matsumoto, Akiko Ebata, Miku Sato, Narumi Harada-Shoji, Hiroshi Tada, Minoru Miyashita, Takanori Ishida, Masamitsu Maekawa, Yoshihisa Tomioka, Nariyasu Mano.

  The enzymatic activity of cytochrome P450 2D6 (CYP2D6) exhibits substantial interindividual variation, which influences drug efficacy and the risk of adverse drug reactions to various therapeutics. While current protocols for CYP2D6 genotyping or CYP2D6 phenotyping provide useful information, less invasive and simpler tools are desirable for evaluating interindividual functional variety. Recently, solanidine (SND) and its metabolites have been proposed as biomarkers of CYP2D6 activity. In this study, we established a validated analytical method to quantify SND and its metabolites. Moreover, their utility in predicting CYP2D6 activity in the Japanese population was assessed. We synthesized and structurally confirmed 4-hydroxysolanidine (4-OHSND) and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of SND, 4-OHSND, and 3,4-seco-solanidine-3,4-dioic acid (SSDA). This method was applied to plasma samples from 94 participants to investigate the CYP2D6 genotypes. The 4-OHSND to SND and SSDA to SND ratios showed substantial differences corresponding to the predicted CYP2D6 phenotype groups, with a marked reduction in poor metabolizers (PMs). Receiver operating characteristic analyses demonstrated complete discrimination of PMs (AUC = 1.00). In contrast, weak positive correlations were identified between SND biomarkers and certain tamoxifen metabolic ratios. These findings confirm the structural identity and quantitative detectability of 4-OHSND and support its potential as a practical biomarker for CYP2D6 PM detection. Although validation in larger and more diverse cohorts remains necessary, diet-derived biomarkers may represent a minimally invasive alternative to genotyping or probe-based approaches and may contribute to the broad implementation of phenotype-guided personalized medicine.

Keywords:  4‐hydroxysolanidine; CYP2D6; LC–MS/MS; phenotyping; poor metabolizer; solanidine

DOI:  https://doi.org/10.1111/cts.70668
J Food Drug Anal. 2026 Jun 01. 34(1): 45-54

Advanced technologies of single-cell metabolomics unveiling cellular metabolic heterogeneity for biological and biomedical research.

Shang-Che Kuo, Ting-Wei Tsao, I-Fan Wang, Ying-Lan Chen.

Metabolomics provides direct insights into cellular physiology, yet it faces greater analytical challenges compared to genomics and transcriptomics due to the chemical diversity, instability, and environmental sensitivity of metabolites. Conventional bulk metabolomics averages signals across cell populations, thereby masking cellular heterogeneity critical for understanding disease mechanisms. Recent breakthroughs in single-cell metabolomics (SCM), driven by advances in mass spectrometry, microfluidics, isotope tracing, and spatial omics, have enabled the detection of metabolic diversity at unprecedented resolution. SCM has uncovered cell-type-specific biomarkers, revealed metabolic reprogramming in cancer and immunity and revealed disease progression. These studies highlight SCM's transformative potential in biomarker discovery, clinical diagnostics, and precision medicine. Despite rapid progress, SCM remains limited by low metabolite abundance, instability during cell handling, lack of standardized quantification methods, and challenges in integrative multi-omics analysis. Future developments will require innovations to improve sensitivity and spatial resolution, establish cross-laboratory quality control frameworks, and apply artificial intelligence for data interpretation. With continued technological convergence, SCM is poised to evolve from a niche research tool into a cornerstone platform for biological and biomedical research.

DOI: https://doi.org/10.38212/2224-6614.3590
Drug Metab Bioanal. 2026 ;19(1): 51-67

Study on the Forced Degradation Behaviour of Oteseconazole and Characterization of Its Degradants by LC-MS/MS.

Meera Devi Mazhavancheril, K G Baheti.

   INTRODUCTION: Oteseconazole is a novel tetrazole antifungal agent used to treat recurrent Vulvovaginal Candidiasis in women. Its mechanism of action is through the inhibition of cytochrome P450(CYP)51, thereby affecting the development and integrity of the fungal cell membrane. The main objective of the present work is to investigate the forced degradation behaviour of Oteseconazole, identify its degradants, and propose the fragmentation pathways of the degradants.
METHODS: Good chromatographic separation of Oteseconazole was achieved using an HPLC System equipped with a PDA detector, and an X-Bridge Phenyl column (150 x 4.6mm, 5μm) with a mobile phase comprising acetonitrile and trifluoroacetic acid buffer (50:50) run in isocratic mode at 268 nm. For mass analysis, the HPLC system was connected to a SCIEX QTRAP 5500 mass spectrometer, operated in positive ion electrospray ionization interface mode.
RESULTS: An accurate, specific, and time-efficient stability-indicating RP-HPLC method was developed for the estimation of Oteseconazole and its degradation products. The developed method was validated with Linearity in a range of 1.25μg/ mL - 7.5 μg/mL, accuracy (%RSD 0.06), system precision (%RSD 0.256), method precision (% RSD 0.49), LOD(0.3μg/mL), and LOQ(1μg/mL). Upon forced degradation studies according to ICH guidelines, Oteseconazole was found to be stable in photolytic, hydrolytic, and thermal conditions but degraded readily in acidic, alkaline, peroxide, and reduction environments.
DISCUSSION: The developed method was robust and can be used in routine analysis to quantify Oteseconazole. The degradation behaviour of Oteseconazole was studied by performing forced degradation studies according to the ICH guidelines, and it was found to be stable in photolytic, hydrolytic, and thermal conditions, but degraded more readily in acidic, alkaline, peroxide, and reductive environments. The degradation products were characterized by LC-MS/MS, and their fragmentation pathways were proposed.
CONCLUSION: Oteseconazole remained stable in photolytic, hydrolytic, and thermal conditions, but showed significant degradation in acidic, alkaline, peroxide, and reductive environments. The degradation products were characterized by LC-MS/MS and characterized as 5-(4-chlorophenyl)-2-(2- (2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)propyl)pyridine (acid impurity, DP1), sodium 4-(6-(2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)propyl)pyridin-3-yl)phenolate (alkali impurity, DP2), 2-(2-(2,4-difluorophenyl)-1,1-difluroropropyl)-5-(4-hydroperoxyphenyl)pyridine (peroxide impurity, DP3) and 4-(6-2(2,4-difluorophenyl)-1,1-difluoro-2-hydroxy-3-(1H-tetrazol-1- yl)propyl)pyridin-3-yl)phenyl hydrogen sulfate (reduction impurity, DP4), and their fragmentation pathways were proposed. The developed method was accurate, specific, less time-consuming, and could be used in routine analysis for the quantification of Oteseconazole.

Keywords:  ICH; LC-MS/MS; Oteseconazole; RP-HPLC; degradation; isocratic; validation

DOI:  https://doi.org/10.2174/0118723128392142250630064916
J Chromatogr A. 2026 Jul 17. pii: S0021-9673(26)00607-2. [Epub ahead of print]1785 467279

Broad-spectrum analytical method for the simultaneous determination of multiple classes of urinary volatile organic compound metabolites.

Tian Qiu, Xiaohong Yu, Jingyu Wang, Xue Bai, Qi Sun, Hongyang Cui, Song Tang, Yifu Lu, Xiaoming Shi.

  Prolonged exposure to volatile organic compounds (VOCs), even at low concentrations, is associated with a range of adverse health effects. The chemical diversity of VOCs results in highly variable biomarker profiles, underscoring the urgent need for comprehensive and efficient analytical strategies to assess urinary VOC metabolites (mVOCs). In this study, we developed a single-run method for the simultaneous quantification of 18 mVOCs with markedly different physicochemical properties, including acidic mercapturic or carboxylic acids, and basic pyrrolidine alkaloids. The extraction workflow was systematically designed based on the acid-base characteristics of the target analytes. Urine samples were spiked with isotope-labeled internal standards, acidified, and loaded onto an Oasis HLB plate. The acidic analytes were collected in eluate, while the basic analytes were collected in the breakthrough, which was later alkalized and re-extracted to recover. Both fractions were combined and analyzed by LC-MS/MS using electrospray ionization (ESI) and multiple reaction monitoring. Notably, carboxyl-containing metabolites were monitored under negative ESI, while pyrrolidine alkaloids were detected under positive ESI within the same chromatographic run. The method exhibited excellent performance, with detection limits ranging from 0.01 μg/L to 2.77 μg/L. Quality control results for ClinChek® reference materials fell within certified ranges, and the method successfully passed the German External Quality Assessment Scheme (G-EQUAS) proficiency testing. Greenness assessments indicated satisfactory eco-friendliness and high applicability. The validated method was applied to the China National Human Biomonitoring Program (CNHBM), revealing significant differences in mVOC concentrations across subgroups stratified by different population types.

Keywords:  Biomonitoring; Greenness assessment; Human urine; Liquid chromatography-tandem mass spectrometry; Volatile organic compound metabolites

DOI:  https://doi.org/10.1016/j.chroma.2026.467279
Anal Chem. 2026 Jul 20.

CSU-EP: Contrastive Learning for Unifying Experimental and Predicted EI-MS Spectra.

Ting Xie, Yaoyu Zhuang, Jiongyu Wang, Hailiang Zhang, Chuanxiu Yu, Zhimin Zhang, Hongmei Lu.

Spectral library matching is the most common method for compound identification in mass spectrometry (MS), but it is limited by the coverage of experimental libraries. In-silico libraries can expand the searchable chemical space, yet their utility is hindered by the experiment-to-prediction gap arising from the discrepancies between experimental and predicted spectra. Here, we propose CSU-EP, a Contrastive spectral unification framework that bridges this gap with a two-stage training strategy. First, it is self-supervisedly pretrained on 1,883,697 predicted electron ionization (EI) mass spectra using masked peak prediction. Second, it is fine-tuned via contrastive learning to unify representations of paired experimental and predicted spectra. Using this fine-tuned encoder, we assemble 2.24 million NEIMS-predicted spectra into a spectral embedding database (CSU-EP-DB). CSU-EP achieves a Recall@1 of 47.10% on the NIST replib benchmark when searching against CSU-EP-DB, outperforming LLM4MS by 7.28% and FastEI by 12.47%. It also demonstrates excellent ability to identify compounds absent from experimental libraries. In a plasma metabolomics application, incorporating a molecular weight filter boosts Recall@5 to 92.86%. To ensure broad accessibility, a user-friendly web server is deployed. By effectively bridging the experiment-to-prediction gap, CSU-EP establishes a new paradigm for reliable, scalable compound identification using in-silico libraries. The source code, models, and CSU-EP web server are accessible at https://github.com/tingxiecsu/CSU-EP.

DOI: https://doi.org/10.1021/acs.analchem.6c01233