bims-metlip 2026-05-10 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2026–05–10
37 papers selected by
Sofia Costa, Matterworks

Latest Developments in Mass Spectrometry-Based Techniques for Metabolomics Analysis.
Development and validation of HILIC-MS/MS method for the analysis of glutathione metabolism in cell cultures.
[Determination of 31 perfluoroalkyl and polyfluoroalkyl substances in water by ultra performance liquid chromatography-triple quadrupole mass spectrometry combined with direct injection].
In-depth phospholipid profiling of lymphoma cells by HPLC coupled with trapped ion mobility spectrometry and high-resolution tandem mass spectrometry.
Impact of Selected Pre-Analytical and Analytical Factors on Untargeted Salivary Metabolomics.
Comprehensive Quantitative Urinary Steroid Profiling of 29 Steroids Using Liquid Chromatography-Tandem Mass Spectrometry.
Evaluation of MassFrontier, MetFrag, MS-FINDER, and SIRIUS for Metabolite Annotation Using an Experimental LC-HRMS Dataset.
Protocol for constructing correlation-based molecular networks from large-scale untargeted metabolomics data.
Data Analysis in Extreme Resolution Mass Spectrometry Untargeted Metabolomics.
Metabolomics: Fundamentals, Methods, Analysis, Limits, and Recommendations.
Separative Techniques Coupled to Mass Spectrometry for Metabolomic Analysis.
A rapid GC-MS method for the simultaneous determination of bupropion and hydroxybupropion in plasma using SALLME and microwave-assisted derivatization.
The Multifaceted Challenge of Normalizing Mass Spectrometry-Based Metabolomics Data.
Scout-triggered MRM from untargeted data: An R-based strategy for multiplexed targeted metabolomics.
A sensitive LC‑MS/MS method for the simultaneous quantification of hexosylceramides and hexosylsphingosines, their precursors and metabolites in cells, plasma, and tissue homogenates.
Rapid Quantification of Ceftobiprole in Human Plasma and Cerebrospinal Fluid by LC-MS/MS and Its Application in Patients with Central Nervous System Infections.
Development and Validation of an SPE-LC-MS Method for the Determination of Epirubicin, Olaparib and Ribociclib in Human Serum.
Development and validation of UHPLC-MS/MS methods for determination of piperaquine in quantitative dried blood samples and microvolume plasma samples.
UHPLC-MS/MS Method for the Simultaneous Quantification of 12 Antiretroviral Drugs in Human Plasma Using Dried Sample Spot Devices: Development, Validation, and Stability Evaluation.
High-Throughput Determination of 210 Pesticide Residues in Gherkins by QuEChERS Coupled with LC-MS/MS and GC-MS/MS.
Development and validation of a semi-automated hybrid immunocapture liquid chromatography-mass spectrometry method to quantify LALA-mutated biotherapeutics in mouse plasma.
Live-Cell Derivatization Enables High-Throughput Analysis of Thiols with CyESI-MS.
Improving Isotope Ratio Accuracy in Metabolic Labeling Using Orbitrap Mass Spectrometry: A Machine Learning Correction Algorithm for Metabolic Flux Analysis.
Mass Spectrometry Based Multi-Omics Integration: Analytical Methods and Biological Applications.
Quantification of nirmatrelvir concentrations in rat plasma, cerebrospinal fluid, and peripheral blood mononuclear cell extracts using LC-MS/MS.
From Mythology to Methodology: Untargeted Single-Cell Metabolomics in R with MeDUSA.
A Unified Bayesian Framework for Cross-Technology Collision Cross Section Postcalibration Correction and Multilaboratory Harmonization.
Multi-Laboratory Assessment Reveals Variable Ion Species Profiles in Electrospray Ionization Mass Spectrometry.
Development and validation of a new derivatization-based quantitative analysis of multi-components by single marker strategy for simultaneous quantification of biogenic amines in fermented wine.
Analysis of weight-loss compounds in health supplements by ultrasonic extraction, dispersive sorbent cleanup, and liquid chromatography tandem mass spectrometry quantification.
Critical Evaluation of Analytical Strategies for the Quantification of Procaine and Its Metabolites Across Diverse Matrices.
A novel green QbD-optimized RP-HPLC bioanalytical method for the simultaneous determination of doxorubicin and rifampicin in human plasma.
From Columns to Clouds: Emerging Interfaces and Ion Sources for Coupling Analytical Separations With Mass Spectrometry.
[Determination of perfluorinated compounds， antibiotics and pesticides in drinking water by automated solid phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry].
[Determination of six whitening ingredients in cosmetics by ultra performance liquid chromatography coupled with photodiode array detector and corona charged aerosol detector].
Comprehensive Impurity and Degradation Profiling of Iptacopan Using a White-Green Stability-Indicating HPLC Method Coupled With LC-MS/MS and In Silico Toxicity Evaluation.
Feasibility verification of fingerstick capillary microsampling replacing venipuncture for therapeutic drug monitoring of 12 antibiotics using a rapid LC-MS/MS assay.

Adv Exp Med Biol. 2026 ;1504 69-93

Latest Developments in Mass Spectrometry-Based Techniques for Metabolomics Analysis.

Marisa Maia, Jasmine Hertzog, Vincent Carré.

  Metabolomics is one of the most recent OMICs sciences and considered one of the most complex. It aims to provide a comprehensive study of small molecules (metabolites) in a biological system to obtain insights of cellular processes and metabolic networks. For such purpose, several advanced analytical techniques such as mass spectrometry (MS) are employed to detect, identify, and even quantify metabolites.In this book chapter, we present the two main approaches (targeted and untargeted) used in metabolomics, as well as their individual advantages and limitations and their complementary strengths. As far as MS analytical techniques are concerned, both the various mass spectrometers and ionization sources, currently used for studying metabolites, are discussed, as well as the field of application linked to their performance.Lastly, recent advances made in exploring the different organisms through single-cell metabolomics as well as metabolomics data integration with other OMIC sciences are also discussed.

Keywords:  Ionization sources; Mass analyzers; Mass spectrometry imaging; Metabolomics; Targeted and untargeted analysis

DOI:  https://doi.org/10.1007/978-3-032-18966-0_4
Anal Bioanal Chem. 2026 May 07.

Development and validation of HILIC-MS/MS method for the analysis of glutathione metabolism in cell cultures.

Miroslav Kubát, Erika Roušarová, Tomáš Roušar, Petr Česla.

  Glutathione is a critical intracellular antioxidant that neutralizes reactive oxygen species and participates in detoxification. The ratio of its two forms, the reduced and disulfide, serves as an indicator of cellular oxidative stress associated with both acute and chronic disorders. Monitoring intracellular levels of glutathione and thiols involved in its metabolism is important for the proper characterization of cellular injury. However, current analytical methods often require tedious chemical derivatization, lack adequate retention and selectivity for highly polar analytes, or suffer from severe matrix effects when profiling the broader metabolic pathway. In this study, a robust hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and optimized for the comprehensive analysis of glutathione metabolism in cell samples without chemical derivatization. The method focused on the label-free quantitation of 21 key metabolites, encompassing intact glutathione and other thiols, their oxidized forms, precursor amino acids, and related sulfur-containing compounds. Chromatographic performance was systematically investigated in HILIC mode using sulfobetaine zwitterionic stationary phase. The final method employed 0.05% difluoroacetic acid in the mobile phase and 5% 5-sulfosalicylic acid for sample preparation, ensuring efficient protein precipitation, stabilization of thiols, and compatibility with electrospray ionization. The method demonstrated high analytical performance, with intra- and inter-day precision (≤5%) and accuracy (≤15%) for all target analytes. Application to A549 lung cancer cells incubated with CdCl₂ and cisplatin for 24 and 48 h revealed significant glutathione depletion and multiple metabolic alterations, including elevated γ-glutamylcysteine levels.

Keywords:  Glutathione; High-performance liquid chromatography; Hydrophilic interaction liquid chromatography; Oxidative stress; Tandem mass spectrometry; Thiols

DOI:  https://doi.org/10.1007/s00216-026-06530-1
Se Pu. 2026 May 08. 44(5): 533-546

[Determination of 31 perfluoroalkyl and polyfluoroalkyl substances in water by ultra performance liquid chromatography-triple quadrupole mass spectrometry combined with direct injection].

Chenglong Yang, Pengfei Li, Yingying Zhang, Lingjia Wang, Minmin Hou, Yali Shi, Yaqi Cai.

  Perfluoroalkyl and polyfluoroalkyl substances （PFAS） possess desirable properties， including hydrophobicity， oleophobicity， surface activity， and thermal and chemical stability. Their extensive production and widespread application have resulted in the pervasive presence of PFAS in diverse environmental media. However， accumulating evidence indicates that PFAS are persistent， capable of long-range transport， bioaccumulative， and toxic； consequently， their adverse effects on ecosystems and humans are of widespread concern. Aquatic environments serve as a major transport pathway and contamination route for PFAS， making accurate measurement of PFAS levels in water crucial for assessing associated environmental and health risks. However， accurate quantification requires multi-step procedures， including sample filtration， enrichment， nitrogen blow-down concentration， and reconstitution， such as solid-phase extraction （SPE） and accelerated solvent extraction （ASE）. These methods are often labor-intensive and time-consuming. Although research on fully automated SPE technology is increasing， it necessitates installation of online SPE systems， which entail high costs and may present limitations in sample throughput per run. With continuous advancements in mass spectrometry， instrumental sensitivity has improved considerably， making direct injection of water samples for multi-analyte analysis technically feasible. However， reports on the use of direct injection methods for detecting PFAS in water remain limited， and the number of target analytes covered in such studies is relatively small. In this study， a direct injection-ultra performance liquid chromatography-triple quadrupole mass spectrometry （UPLC-MS/MS） method was developed for the determination of 31 PFAS in water. To optimize the chromatographic separation， enhance the detection sensitivity of target analytes， and minimize undesirable adsorption losses， the method was meticulously optimized with respect to solvent selection， injection volume， and syringe filter type. Our method involves the following procedure： 0.5 mL of water is aliquoted， mixed with 0.5 mL of methanol spiked with 2 ng of internal standard， and filtered through a 0.22 μm polypropylene membrane. The PFAS were analyzed by UPLC-MS/MS with an injection volume of 35 µL. The analytes were ionized in electrospray ionization negative mode （ESI-） with scheduled multiple-reaction monitoring （sMRM）. The MS parameters， including precursor and product ions， collision energy， and declustering voltage were optimized. Through optimization of the analytical column and mobile phases， the analytes were separated on an RSLC 120 C18 column with a gradient of methanol and 5 mmol/L ammonium acetate aqueous solution as the mobile phase in a gradient elution program. The results were quantified by the internal standard method. The method demonstrated excellent linearity （R²>0.994） across a defined concentration range. The limits of detection （LODs） and quantification （LOQs） were 0.007 1-3.0 ng/L and 0.024-10 ng/L， respectively. Recoveries at spiked levels of 2， 10， and 500 ng/L ranged from 67.2% to 130.2%， with relative standard deviations （RSDs） of 0.30% to 18%. To quantify the effective equivalence between the enrichment efficiency of SPE and the sensitivity of direct injection methods， a comparative analysis of analyte recovery rates was performed for both approaches. Furthermore， for long-chain PFAS， direct injection demonstrated consistent and favorable recovery performance. The method was applied to analyze PFAS in groundwater samples. The results showed that 24 PFAS were detectable with the total PFAS content （∑PFAS） ranging from 20.6 to 521 ng/L， with perfluorooctanoic acid （PFOA） and perfluorobutanoic acid （PFBA） being the primary pollutants. This approach is simple， rapid， highly sensitive， and provides broad coverage of target analytes， making it suitable for the quantitative analysis of PFAS in urban groundwater. It offers an efficient and reliable technical solution for determining trace-level PFAS in environmental water samples.

Keywords:  direct injection; groundwater; perfluoroalkyl and polyfluoroalkyl substances; ultra performance liquid chromatography-triple quadrupole mass spectrometry

DOI:  https://doi.org/10.3724/SP.J.1123.2025.09012
J Chromatogr A. 2026 Apr 21. pii: S0021-9673(26)00353-5. [Epub ahead of print]1779 467023

In-depth phospholipid profiling of lymphoma cells by HPLC coupled with trapped ion mobility spectrometry and high-resolution tandem mass spectrometry.

Dominik Wieland, Anja Schmitt, Stephan Hailfinger, Heiko Hayen.

  Lipids exhibit extensive molecular diversity and structural complexity, which poses major analytical challenges for comprehensive lipidomic profiling. Phospholipids, in particular, display extensive structural diversity and isomerism. Given the limited lipidomic data available for lymphoma cells, this work focuses on comprehensive phospholipid screening, which inherently requires the characterization of isomeric species, including plasmalogens that have been implicated in oxidative stress and ferroptosis-related cell death. Therefore, we present an efficient isomer-selective workflow based on reversed-phase liquid chromatography (RPLC) coupled to trapped ion mobility spectrometry (TIMS) and high-resolution tandem mass spectrometry (HR-MS/MS). High-confidence structural lipid annotation is achieved through the integrated evaluation of chromatographic retention time (tR), exact mass-to-charge ratio (m/z), collision cross section (CCS) and mobility-resolved MS/MS data. Applied to human lymphoma cell lipid extracts, the workflow enabled confident identification of 263 individual lipid species spanning 10 phospholipid and 2 sphingolipid subclasses, including the resolution of 63 isomeric species at the fatty-acyl compositional level. The multidimensional approach allowed partial discrimination of fatty-acyl compositional, sn- and double bond positional isomers. Notably, characteristic deviations in both retention time and ion mobility were observed for plasmalogens relative to alkyl-ether linked phospholipids, reflecting the unique physicochemical properties of the vinyl-ether linkage. These systematic offsets enabled confident plasmalogen assignment in representative cases, supported by authentic standards, co-chromatograms and mobility-resolved fragmentation data. Collectively, this streamlined analytical platform markedly expands phospholipidome coverage and provides enhanced structural resolution of complex lipid mixtures.

Keywords:  Ether-lipids; Ion mobility; Isomers; LC-MS/MS; Lymphoma cells; Phospholipids; Plasmalogens

DOI:  https://doi.org/10.1016/j.chroma.2026.467023
Int J Mol Sci. 2026 Apr 08. pii: 3345. [Epub ahead of print]27(8):

Impact of Selected Pre-Analytical and Analytical Factors on Untargeted Salivary Metabolomics.

Sylwia Michorowska, Agnieszka Zięba, Dorota Olczak-Kowalczyk, Joanna Giebułtowicz.

  With the growing interest in personalized medicine, alternative biological matrices to blood are increasingly explored as sources of diagnostic information. Saliva has emerged as a promising diagnostic matrix due to its non-invasive collection, suitability for home sampling, and minimal requirements for personnel training. Numerous studies have demonstrated the presence of metabolites in saliva that enable disease diagnosis and monitoring. However, the influence of pre-analytical and analytical factors on salivary metabolomics outcomes remains insufficiently characterized. In this study, we investigated factors potentially affecting the number and abundance of detected metabolites in untargeted salivary metabolomics using liquid chromatography coupled with mass spectrometry (LC-MS). The impact of chromatographic column type, extraction protocol, and saliva type (stimulated versus resting) was evaluated. Additionally, the effect of swab type on analyte recovery was assessed. The use of a synthetic swab for saliva collection yielded results most comparable to those obtained without swabs, for both resting and stimulated saliva samples, indicating minimal pre-analytical interference. The greatest metabolite coverage was obtained using ACN:MeOH (1:1, v/v), with a ZIC-HILIC column for polar metabolites and a C18 column for non-polar metabolite separation. These findings demonstrate that swab type, chromatographic column, extraction solvent, and saliva type critically shape metabolite coverage in untargeted salivary metabolomics. Importantly, the distinct metabolic profiles of resting and stimulated saliva suggest that these matrices may provide complementary clinical insights, underscoring the need for saliva type selection tailored to specific diagnostic and biomarker discovery objectives.

Keywords:  mass spectrometry; method development; pre-analytical factors; saliva; untargeted metabolomics

DOI:  https://doi.org/10.3390/ijms27083345
Anal Sci Adv. 2026 Jun;7 e70087

Comprehensive Quantitative Urinary Steroid Profiling of 29 Steroids Using Liquid Chromatography-Tandem Mass Spectrometry.

Joshua T Bain, Fozia Shaheen, Alessandro Prete, Lorna C Gilligan, Angela E Taylor.

  Steroids are critical for numerous physiological processes; disruption in their metabolism is associated with numerous endocrine disorders. Steroid quantification is essential to improve the understanding and diagnosis of these pathologies. Historically, urinary steroid profiling has been performed using low-throughput gas chromatography mass spectrometry (GC-MS), providing holistic coverage of steroid classes with low cross-reactivity. Here, we translate our previous GC-MS urinary steroid profile to a liquid chromatography tandem-MS (LC-MS/MS) platform, offering a validated, comprehensive overview of steroid metabolism with comparatively low sample preparation times and increased throughput. Urinary steroids were enzymatically hydrolysed and extracted via C18 solid-phase extraction. Quantification was conducted using a triple quadrupole mass spectrometer. Chromatographic separation of 27 analytes was completed in 16 min using a C18-T3 column. Due to chromatographic co-elution of tetrahydrocortisol and 5α-tetrahydrocortisol, a second injection was required on a BEH-C18 column for their separation. Lower limit of quantification (LLOQ) ranged from 2 to 20 ng/mL, witha ccuracy (bias) ranging from -18.7% to 19.9%, and precision (percentage coefficient of variation [%CV]) ranging from 4.0% to 18.6%. Matrix effects remained within the ideal range <±15% for all steroids. Recovery ranged from 76% to 103%, and intra- and inter-assay imprecision (CV) ranged from 0.8% to 14.9%. In 40 healthy volunteers, most analytes were detected above the LLOQ in over 95% of samples, although tetrahydro-aldosterone (85%), 5-pregenediol (68%), and pregnanetriol-one (59%) demonstrated lower quantification rates. Diurnal and sex-based variations were observed, with excretion levels significantly higher during daytime and in males. This robust, high-throughput LC-MS/MS method facilitates the simultaneous quantification of multiple steroid classes, enhancing its utility for clinical and research applications in endocrine science.

Keywords:  LC‐MS/MS; androgens; glucocorticoids; mineralocorticoids; steroid precursors; steroid profiling; urine

DOI:  https://doi.org/10.1002/ansa.70087
Biomedicines. 2026 Apr 10. pii: 872. [Epub ahead of print]14(4):

Evaluation of MassFrontier, MetFrag, MS-FINDER, and SIRIUS for Metabolite Annotation Using an Experimental LC-HRMS Dataset.

Dmitrii A Leonov, Irina A Mednova, Alexander A Chernonosov.

  Background: Untargeted metabolomics enables comprehensive profiling of biological systems, but accurate metabolite annotation remains a critical bottleneck due to incomplete spectral libraries and structural isomerism. The use of in silico annotation tools can increase the coverage of annotated compounds, but it remains unclear whether these tools, in the absence of reference standards, can reliably annotate real-world experimental LC-HRMS data and whether they are sufficient for this task. Methods: This study assesses the performance and limitations of four widely used in silico structure prediction tools (MassFrontier, MetFrag, MS-FINDER, and SIRIUS/CSI:FingerID) when applied to an experimentally acquired feature set previously used to differentiate patients with depressive disorders from healthy controls. To ensure uniform evaluation across tools under realistic but optimized conditions, the quality of MS/MS data was improved using a parallel reaction monitoring method, allowing acquisition of interpretable fragmentation spectra for 26 of the 28 detected features. Results: For most features, all tools were able to suggest structure candidates. However, none of the tools proved sufficient as a standalone solution for reliable metabolite annotation. Due to their different algorithms, each tool had strengths and weaknesses in fragmentation interpretation, candidate generation, and ranking, resulting in incomplete or inconsistent annotations. While the combined application of all four tools provided a substantial improvement in putative annotation over conventional spectral library matching, the in silico structure prediction tools often prioritized chemically implausible, biologically irrelevant, or artifactual candidates. Consequently, manual expert evaluation was required to assess the chemical plausibility and biological relevance of the proposed structures. This ultimately reduced the number of biologically plausible metabolites putatively associated with disease to ten. Conclusions: Overall, these results demonstrate that existing in silico annotation tools can substantially support the annotation of experimental metabolomics data, but are insufficient on their own. Reliable identification of metabolites in complex biological matrices still depends on high-quality MS/MS data acquisition, the combined use of complementary tools, and mandatory post-annotation expert curation.

Keywords:  MS-FINDER; MassFrontier; MetFrag; PRM; SIRIUS; biomarker discovery; in silico annotation; mass spectrometry; untargeted metabolomics

DOI:  https://doi.org/10.3390/biomedicines14040872
bioRxiv. 2026 Apr 21. pii: 2025.04.26.649581. [Epub ahead of print]

Protocol for constructing correlation-based molecular networks from large-scale untargeted metabolomics data.

Huang Lin, Lijun Zhang, Ali Lotfi, Alan Jarmusch, Iris Lee, Adam Kim, James T Morton, Alexander Aksenov.

This protocol describes a computational approach for constructing correlation-based molecular networks from untargeted metabolomics data using MetVAE, a variational autoencoder-based framework. Complementing spectral similarity networks, it captures functional relationships re-flected in cross-sample correlations. The workflow imports metabolomics features and sample metadata, adjusts for compositionality, missingness, confounding, and high-dimensionality, esti-mates sparse metabolite correlations, and exports GraphML files for network visualization. In a hepatocellular carcinoma mouse model, it links lipid classes in high-fat-diet animals, suggesting an endogenous "auto-brewery" route to lipotoxic metabolites.

DOI: https://doi.org/10.1101/2025.04.26.649581
Adv Exp Med Biol. 2026 ;1504 247-269

Data Analysis in Extreme Resolution Mass Spectrometry Untargeted Metabolomics.

Marta Sousa Silva, Francisco Traquete, João Luz, António E N Ferreira, Carlos Cordeiro.

  Mass spectrometry (MS)-based metabolomics is a powerful tool for understanding the complexity of biochemical processes and to identify biomarkers across diverse biological systems. The vast amount of data generated by extreme resolution mass spectrometers poses significant data processing challenges, requiring robust computational approaches and workflows for meaningful data interpretation. This chapter provides a comprehensive overview of current methodologies in MS-based metabolomics data analysis, with a focus on data preprocessing and pretreatment, m/z extraction and annotation, univariate and multivariate statistical approaches, as well as data visualization. We discuss key considerations for ensuring data quality and the growing role of bioinformatics in pathway analysis and metabolite identification. We highlight the transforming role of extreme resolution and mass accuracy enabled by FT-ICR mass spectrometers, and finally, we explore emerging trends, including artificial intelligence-driven insights and real-time data processing, to guide future developments in this rapidly evolving field.

Keywords:  Compound identification; Data processing and treatment; FT-ICR-MS; Untargeted metabolomics

DOI:  https://doi.org/10.1007/978-3-032-18966-0_12
Adv Exp Med Biol. 2026 ;1504 119-144

Metabolomics: Fundamentals, Methods, Analysis, Limits, and Recommendations.

Ayush Praveen, Tripti Nair, Biplab Singha, Sumit Murmu, Murugesh Padmanarayana, Aditya Upadhyay, Yogita Soni, Vijay Soni.

  Metabolomics has emerged as a powerful discipline for characterizing the small molecules that define cellular physiology, environmental responses, and disease states. As technologies advance, researchers face an expanding landscape of analytical platforms, data-processing strategies, and integrative approaches that require clear guidance for effective application. This chapter was written to provide a comprehensive and accessible resource for students, clinicians, and researchers entering or advancing in the field. We outline the fundamentals of metabolomics, describe major analytical methodologies-including MS, NMR, chromatography, and imaging-and summarize key considerations for experimental design, data preprocessing, statistical analysis, and functional interpretation. We also address current challenges related to metabolite identification, reproducibility, and multi-omic integration, and highlight emerging innovations such as stable-isotope tracing, spatial metabolomics, and AI-driven analytics. Together, these elements offer a detailed roadmap for conducting robust, reproducible, and insightful metabolomic studies.

Keywords:  Capillary electrophoresis; Chemoinformatic; Chromatography; Cross-Omics; Heteroscedasticity; Mass analyzing; Metabolite imaging; Metabolome; Microbiome; Network modeling; Small molecules

DOI:  https://doi.org/10.1007/978-3-032-18966-0_6
Adv Exp Med Biol. 2026 ;1504 95-118

Separative Techniques Coupled to Mass Spectrometry for Metabolomic Analysis.

Jasmine Hertzog, Marisa Maia, Vincent Carré.

  Advanced analytical techniques are required to decipher metabolome complexity. The previous chapter ("Latest Developments in Mass Spectrometry-Based Techniques for Metabolomics Analysis") shows general aspect of mass spectrometry techniques for the detection, identification, and quantification of metabolites by target and non-targeted approaches. It also points out that, in some instances, the sole direct analysis by mass spectrometry is a limitation for deciphering the high molecular complexity of the metabolome. Therefore, upstream separative methods such as chromatography, capillary electrophoresis, or ion mobility can be an effective solution. Indeed, these techniques reduce the ion suppression effects, separate some isomers, and increase the capacity measurement for metabolite identification and quantification. This chapter focuses on the separative methods most frequently used in metabolomic studies. Their principles and some examples are addressed along with their coupling with MS.Lastly, analytical improvements and recent advances in MS hyphenated approaches for metabolomic study are also discussed with multidimensional analysis.

Keywords:  Capillary electrophoresis; Chromatography; Ion mobility; Metabolomics; Separative techniques

DOI:  https://doi.org/10.1007/978-3-032-18966-0_5
J Chromatogr A. 2026 Apr 28. pii: S0021-9673(26)00379-1. [Epub ahead of print]1779 467049

A rapid GC-MS method for the simultaneous determination of bupropion and hydroxybupropion in plasma using SALLME and microwave-assisted derivatization.

Merve Neşetoğlu, Selen Al, Neşet Neşetoğlu, Aykut Kul, Olcay Sagirli.

  Bupropion is widely used in the treatment of major depressive disorder and smoking cessation, and its clinical response is largely influenced by its active metabolite, hydroxybupropion. Due to significant pharmacokinetic variability and the therapeutic relevance of hydroxybupropion concentrations, reliable analytical methods suitable for therapeutic drug monitoring (TDM) are required. In this study, a rapid gas chromatography-mass spectrometry (GC-MS) method was developed for the simultaneous determination of bupropion and hydroxybupropion in human plasma using a novel sample preparation strategy termed KUL-SPEED (Kul's Ultra-fast, Low-step Sample Preparation with Efficient Extraction and Derivatization). The method integrates salt-assisted liquid-liquid microextraction (SALLME) with microwave-assisted trimethylsilylation in a unified workflow, eliminating solvent evaporation and reconstitution steps. Sample preparation and derivatization were completed within approximately 5 min, and chromatographic separation was achieved within an 8-minute total run time. Method validation was performed according to ICH M10 guidelines. Calibration was linear over 50-2000 ng/mL for bupropion and 250-2000 ng/mL for hydroxybupropion, with acceptable accuracy, precision, stability, and matrix effect results. The method was successfully applied to patient plasma samples, confirming its applicability for routine TDM. The KUL-SPEED approach may also be adaptable to other derivatizable analytes in biological matrices.

Keywords:  Bupropion; GC–MS; Hydroxybupropion; KUL-SPEED; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.chroma.2026.467049
Anal Chem. 2026 May 04.

The Multifaceted Challenge of Normalizing Mass Spectrometry-Based Metabolomics Data.

Brian Low, Chau Nguyen, Tao Huan.

Normalization is a critical step in metabolomics studies to ensure the quality of metabolomics data, reduce quantitative variability, and enable confident and robust statistical analyses. From an analytical perspective, metabolomics normalization encompasses multiple distinct processes. Broadly, normalization can refer to (1) sample normalization, which mitigates variation due to differences in total metabolite amounts; (2) signal correction, which reduces batch effects, instrumental fluctuations, and retention time drifts during data collection; and (3) statistical transformation and scaling, which prepare data for statistical analyses. Each of these normalization processes addresses unique analytical and bioinformatic needs, but the term "normalization" is often used broadly, leading to confusion in method development, selection, and implementation. Moreover, many well-established normalization algorithms in genomics and proteomics are not always transferable to metabolomics due to differences in analytical workflows and data characteristics. To address these issues, we believe it is crucial to gain a clear understanding of the purpose of each normalization type, its appropriate implementation, and the evaluation criteria. This perspective outlines the key normalization tasks in metabolomics, reviews existing tools, and provides recommendations for their appropriate applications. We also highlight two critical considerations: (1) selecting an appropriate missing value imputation method and (2) establishing strategies to evaluate and compare normalization outcomes. The goal of this work is to provide recommendations for the rigorous development and implementation of normalization techniques in metabolomics, thereby enhancing analytical accuracy and precision, improving data interpretability, and ultimately advancing the biological insights gained from metabolomics studies.

DOI: https://doi.org/10.1021/acs.analchem.6c00292
Talanta. 2026 Apr 27. pii: S0039-9140(26)00521-7. [Epub ahead of print]307 129865

Scout-triggered MRM from untargeted data: An R-based strategy for multiplexed targeted metabolomics.

Rémy De Boni, Guillaume Rossignol, Delphine Arquier, Benoît Joseph, Guy Fournet, Jérôme Lemoine, Jérôme Randon, Sophie Ayciriex, Yohann Clement, Arnaud Salvador.

  The transition from untargeted discovery to targeted validation in metabolomics is a major challenge, often hindered by poor method transferability and retention time (RT) variation between liquid chromatography-mass spectrometry (LC-MS) platforms. To address this, "Scout-MRM Builder," an R-based package for the automated creation of highly multiplexed targeted methods from untargeted high-resolution MS2 data, is presented here. A Scout-Triggered Multiple Reaction Monitoring (StMRM) strategy is employed, using N-Alkylpyridinium-3-Sulfonate (NAPS) standards as dynamic RT markers ("scouts"). A specific list of transitions is triggered by the detection of each scout, ensuring robustness against RT shifts. Ion pairs are automatically extracted, scouts are identified, and ready-to-use StMRM methods, including pseudo-MRM transitions for features lacking fragmentation spectra, are generated. From an untargeted analysis of porcine liver extracts, a single StMRM method monitoring 1312 transitions was generated from 558 features. High reproducibility was demonstrated, with 89.9% of detected transitions exhibiting a relative standard deviation (RSD) below 20%. When applied to a model of liver ischemia-reperfusion injury, results highly comparable to the initial untargeted analysis were obtained. A common core of potential biomarkers was identified, with slightly improved statistical performance. In conclusion, the Scout-MRM Builder provides a powerful framework to bridge the gap between discovery and validation, enabling robust targeted analysis at an untargeted scale through enhanced method transferability and reliability.

Keywords:  Liquid chromatography-mass spectrometry; Liver ischemia-reperfusion; Method development; Scout-triggered MRM; Targeted metabolomics; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.talanta.2026.129865
Anal Bioanal Chem. 2026 May 07.

A sensitive LC‑MS/MS method for the simultaneous quantification of hexosylceramides and hexosylsphingosines, their precursors and metabolites in cells, plasma, and tissue homogenates.

Michele Iannone, Tine Loomans, Sara Gorremans, Elien Grajchen, Celine Deneubourg, Brian Hrupka, Luc Ver Donck, Alexis Bretteville, Diederik Moechars, Rob J Vreeken, Farid Jahouh.

  Hexosylceramides (HexCers) and hexosylsphingosines (HexSphs) are bioactive glycosphingolipids implicated in neurodegenerative diseases such as Parkinson's disease (PD), Gaucher disease, and Krabbe disease. Despite their biological relevance, no existing method enables the simultaneous quantification of both HexCers and HexSphs across multiple biological matrices. In this study, we report the development, validation, and application of a novel LC-MS/MS method capable of quantifying 11 lipid species, including glucosylceramides, galactosylceramides, glucosylsphingosine, galactosylsphingosine, ceramide, lactosylceramide, and sphingosine, in a single chromatographic run. The method integrates optimized liquid-liquid extraction and solid-phase extraction protocols, achieving high recovery and selectivity. Validation was performed according to internal and external (e.g., ICH and FDA guidelines) international guidelines, demonstrating excellent sensitivity, linearity (R2 > 0.99), precision (cv < 15%), and stability across cells, mouse plasma, and brain tissue homogenates. Proof-of-concept applications included wild-type (WT) SKMEL28 cells treated and non-treated with the glucocerebrosidase inhibitor CBE, as well as WT mouse plasma and brain regions (cortex, hippocampus, midbrain, cerebellum), establishing baseline levels for future pharmacokinetic studies. This highly comprehensive method offers a robust analytical platform for sphingolipid profiling in neurodegenerative disease research.

Keywords:  HILIC chromatography; Hexosylceramides; Hexosylsphingosines; LC-MS/MS quantification; Neurodegenerative disease biomarkers; Sphingolipid metabolism

DOI:  https://doi.org/10.1007/s00216-026-06485-3
Molecules. 2026 Apr 10. pii: 1252. [Epub ahead of print]31(8):

Rapid Quantification of Ceftobiprole in Human Plasma and Cerebrospinal Fluid by LC-MS/MS and Its Application in Patients with Central Nervous System Infections.

Sabahat Ablimit, Wanzhen Li, Mengting Chen, Jing Zhang, Nanyang Li, Yaxin Fan, Muyassar Yasen, Mubarak Iminjan, Beining Guo.

  Ceftobiprole is a fifth-generation beta-cephalosporin with high inter-individual pharmacokinetic variability in critically ill patients. However, data on its pharmacokinetics and central nervous system (CNS) penetration are limited. This study developed and validated a rapid LC-MS/MS method for quantifying ceftobiprole in human plasma and CSF. Sample preparation involved protein precipitation of 50 µL aliquots. Analysis used gradient elution on an ACQUITY UPLC® HSS T3 column (2.1 × 100 mm, 1.8 µm) with 0.2% formic acid and acetonitrile and was detected by positive ion electrospray, achieving a 3.5 min run time. The method was linear from 0.100 to 25.0 mg/L in plasma and 0.0500 to 15.0 mg/L in CSF. Intra- and inter-run precision and accuracy were within ±15% at all quality control levels. All validation parameters, including selectivity, matrix effects, recovery, and stability under various conditions, met acceptance criteria. Potential interference from the prodrug ceftobiprole medocaril was evaluated and found to be negligible. The method was successfully applied to samples from three patients, revealing a CSF penetration range of 11.9% to 36.5%. This validated LC-MS/MS method enables simple and rapid quantification of ceftobiprole in plasma and cerebrospinal fluid, filling the gap in data on its CNS penetration and supporting routine drug concentration monitoring in critically ill patients.

Keywords:  ceftobiprole; cerebrospinal fluid; liquid chromatography–tandem mass spectrometry; penetration; plasma

DOI:  https://doi.org/10.3390/molecules31081252
Biomedicines. 2026 Apr 08. pii: 848. [Epub ahead of print]14(4):

Development and Validation of an SPE-LC-MS Method for the Determination of Epirubicin, Olaparib and Ribociclib in Human Serum.

Monica Denisa Elena Popescu, Costel-Valentin Manda, Octavian Croitoru, Daniela-Maria Calucică, Johny Neamțu, Andrei Biță, Amelia Maria Găman, Simona-Daniela Neamțu.

  Background/Objectives: Epirubicin, Olaparib, and Ribociclib are widely used anticancer agents whose serum concentrations exhibit significant inter-individual variability, supporting the need for reliable and robust analytical methods suitable for pharmacokinetic evaluation and therapeutic exposure assessment. Variations in metabolism, drug-drug interactions, organ function, and treatment regimens may substantially influence systemic exposure, highlighting the importance of accurate quantification in clinical practice. This study describes the development and validation of a solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) method for the simultaneous quantification of these drugs in human serum. Methods: Sample preparation was performed using Oasis PRiME HLB® cartridges to ensure efficient clean-up, optimal recovery, and reduced matrix effects. Chromatographic separation was achieved using gradient elution with 0.1% formic acid and acetonitrile on a reversed-phase column, followed by single-quadrupole mass spectrometric (QDa) detection in the selected ion recording mode. The total run time was 13 min, enabling high-throughput analysis. Results: The method demonstrated good linearity (r > 0.997) over the tested concentration ranges, along with adequate selectivity, precision, accuracy, recovery, and stability, fulfilling the ICH M10 guideline validation criteria. No significant carry-over or interference from endogenous compounds was observed. Conclusions: Application to patient samples confirmed reliable performance in real clinical matrices and consistent quantification across different concentration levels. The proposed approach provides a potentially more accessible alternative in laboratories already equipped with LC-MS systems compared to LC-MS/MS platforms and can be applied in pharmacokinetic studies, representing a proof-of-concept for exposure assessment in oncology.

Keywords:  LC-MS; SPE; epirubicin; human serum; olaparib; ribociclib

DOI:  https://doi.org/10.3390/biomedicines14040848
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 May 04. pii: S1570-0232(26)00194-7. [Epub ahead of print]1279 125105

Development and validation of UHPLC-MS/MS methods for determination of piperaquine in quantitative dried blood samples and microvolume plasma samples.

Meiyun Serena He, Sindy He, Michelle Roh, Abel Kakuru, Prasanna Jagannathan, Grant Dorsey, Philip Rosenthal, Francesca Aweeka, Liusheng Huang.

  Piperaquine (PPQ) is an antimalarial drug used in combination with dihydroartemisinin. To support pharmacokinetic studies of PPQ in children and resource-limited settings, we developed assays to determine PPQ in dried blood samples (DBS) collected with Capitainer devices (10 μL blood) and in 10 μL plasma samples. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was employed. DBS samples were soaked with 1% HCl to extract PPQ into solution followed by neutralization with NH4OH and extraction with methyl acetate, while the plasma samples underwent liquid-liquid extraction. The organic layer was dried and reconstituted in acetonitrile-water-formic acid (50:50:0.5, v/v/v) before UHPLC-MS/MS analysis using a pentafluorophenyl reverse-phase column (2.1 × 30 mm, 1.8 μm). Atmospheric pressure chemical ionization in positive mode was used with multiple reaction monitoring of m/z 535 → 288 for PPQ and 541 → 294 for PPQ-d6 as the internal standard. Both analytes eluted at 0.52 min, with a total run time of 2 min per sample. Calibration ranges were 1-500 ng/mL for DBS and 0.5-500 ng/mL for plasma. Recoveries were 54.4-59.0% for DBS and 69.3-82.9% for plasma, with negligible matrix effect and hematocrit effect. DBS samples were stable for at least 8 months at room temperature. Clinical validation with 46 pairs of simultaneously collected DBS and plasma samples demonstrated a strong linear relationship between PPQ concentrations in DBS and plasma (logCDBS = 1.02 x logCplasma + 1.01, R2 = 0.919), with a median (IQR) DBS/plasma ratio of 2.84 (1.97-3.95). The assays will facilitate pharmacokinetic studies in children and resource-limited settings.

Keywords:  Pediatric; Piperaquine; Plasma; Quantitative DBS; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2026.125105
Pharmaceutics. 2026 Apr 21. pii: 513. [Epub ahead of print]18(4):

UHPLC-MS/MS Method for the Simultaneous Quantification of 12 Antiretroviral Drugs in Human Plasma Using Dried Sample Spot Devices: Development, Validation, and Stability Evaluation.

Sara Soloperto, Elisa Martina, Alice Palermiti, Elisa Barnini, Greta Sabbia, Gianluca Bianco, Martina Billi, Camilla Martino, Alessandra Manca, Marco Simiele, Jessica Cusato, Antonio D'Avolio, Amedeo De Nicolò.

  Background/Objectives: In several contexts, Dried Sample Spot Devices (DSSDs) offer a convenient and safe alternative for sampling, storage, and shipment, allowing the transport and storage of biological samples at room temperature, reducing shipment costs and improving access to diagnostics in faraway sites. This can be pivotal for the use of the therapeutic drug monitoring of anti-HIV treatment: therefore, this study aimed to develop and validate a UHPLC-MS/MS method for the simultaneous quantification of 12 antiretroviral drugs, including the recently introduced long-acting agents, in Dry Plasma Spots (DPSs). Methods: First, 100 µL of plasma sample and 100 µL of internal standard solution were spotted on each DSSD. After complete drying, DPSs were added with an acidifying solution (ammonium acetate buffer pH 4), and then, each sample underwent extraction with hexane-dichloromethane 50:50 (v/v). After tumbling, the organic phase was evaporated and reconstituted for injection. An Acquity UPLC HSS T3 1.8 µm, 2.1 × 150 mm column at 50 °C enabled separation, performed using H2O + F.A. 0.05% (phase A) and ACN + F.A. 0.05% (phase B) as the mobile phase in gradient elution mode, for a total run time of 15 min. Results: The method was validated over the clinically relevant concentration ranges. For all quality control levels, accuracies ranged from 98.2% to 114.1%, and intra-day and inter-day RSD values ranged from 2.7% to 9.7% and 5.2% to 13.9%, respectively. All analytes demonstrated satisfactory short- and long-term stability in DPSs, confirming the suitability of shipment and storage at room temperature. Conclusions: The method demonstrated robustness and reproducibility in accordance with FDA and EMA guidelines. It ensures satisfactory accuracy and rapid analysis, supporting its application in clinical practice, including for monitoring the newest long-acting drugs.

Keywords:  antiretrovirals; dried plasma spot; liquid chromatography; long acting; mass spectrometry; microsampling; pharmacokinetics; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/pharmaceutics18040513
Molecules. 2026 Apr 09. pii: 1248. [Epub ahead of print]31(8):

High-Throughput Determination of 210 Pesticide Residues in Gherkins by QuEChERS Coupled with LC-MS/MS and GC-MS/MS.

Mehmet Keklik, Eylem Odabas, Tuba Buyuksirit-Bedir, Ozgur Golge, Miguel Ángel González-Curbelo, Bulent Kabak.

  Pesticide residues represent an important group of chemical contaminants in agricultural commodities and require reliable analytical strategies for accurate monitoring. In this study, a high-throughput analytical workflow was applied for the determination of 210 pesticide residues in gherkins. Sample preparation was performed using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, including extraction followed by dispersive solid-phase extraction clean-up. Residue determination was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). The analytical methods were comprehensively validated in the gherkin matrix in accordance with the SANTE 11312/2021 v2 guidelines. Limits of quantification were ≤0.01 mg kg-1 for all compounds. Recovery values ranged from 75.7% to 113.7%, while precision values remained below 20%, demonstrating satisfactory method accuracy and precision. Expanded measurement uncertainty values ranged between 7.6% and 41.3%, confirming the robustness of the validated analytical workflow. The validated methods were subsequently applied to a large-scale monitoring dataset comprising 905 gherkin samples collected from five major production regions in Türkiye. Pesticide residues were detected in 67.6% of the analysed samples, and 37 different compounds were identified. The most frequently detected pesticides were flonicamid (36.2%) and propamocarb (27.5%). Multi-residue contamination was frequently observed, reflecting complex pesticide application patterns in gherkin cultivation systems. Although chronic exposure estimates remained well below toxicological thresholds for both adults and children, certain exposure scenarios indicated that acute exposure for children may warrant further attention.

Keywords:  chromatographic techniques; food contaminants; method performance evaluation; multi-class pesticides; multi-residue analysis; trace-level determination

DOI:  https://doi.org/10.3390/molecules31081248
Eur J Pharm Sci. 2026 Apr 30. pii: S0928-0987(26)00114-4. [Epub ahead of print] 107540

Development and validation of a semi-automated hybrid immunocapture liquid chromatography-mass spectrometry method to quantify LALA-mutated biotherapeutics in mouse plasma.

Vittoria Papa, Ilse De Salve, Francesco Molinaro, Andrea Di Ianni, Stefania Monge, Rita Mastroianni, Alessandra Ariaudo, Patrizia Tavano, Valeria Castagna, Kyra Cowan, Federico Riccardi Sirtori.

  Biotherapeutic antibodies are increasingly being developed and, various strategies have recently been used to maximize their potential therapeutic efficacy. The crystallizable fragment (Fc) region of therapeutic monoclonal antibodies (mAbs) is often engineered to tailor their effector functions and pharmacokinetic (PK) properties by introducing point mutations. Notably, most of these mutations are in the hinge and constant domains of the heavy chain, which may silence antibody effector functions. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been published to quantify biotherapeutics with a canonical human Fc portion. This work presents a rapid and sensitive hybrid immunocapture liquid chromatography-tandem mass spectrometry (IC-LC-MS/MS) method for quantifying total antibody concentration, specifically targeting the LALA-mutated peptide (L234A/L235A). The sample preparation process, which includes immunocapture, as well as trypsin and Glu-C digestion, is efficiently completed within two days through automation. The developed method was validated according to the ICH M10 guideline and white papers recommendation, focusing on the following parameters - accuracy, precision, dilution linearity, selectivity, stability, recovery- and using a humanized IgG1 LALA-mutated antibody, teplizumab, as analytical standard. The method demonstrated linearity for total antibody detection in mouse plasma samples, with a dynamic range from 150 ng/mL (lower limit of quantification, LLOQ) to 15,000 ng/mL (upper limit of quantitation, ULOQ). All validation parameters tested in mouse plasma met the predefined acceptance criteria, demonstrating the method's reliability and robustness. Additionally, the qualified method was successfully used to characterize the pharmacokinetic profile in mice of an antibody-drug conjugate (ADC-1) containing the LALA mutation in its Fc region. This work provides a valuable foundation for the quantification of new biological entities (NBEs) and antibody-drug conjugates (ADCs) in pharmaceutical development, as it enables the measurement of engineered Fc biotherapeutics using a unique and highly selective peptide, irrespective of the type of biological matrix and even in the presence of other biomolecules of similar IgG isotype.

Keywords:  Drug development; LALA mutation; LC-MS; bioanalysis; mass spectrometry; pharmacokinetics

DOI:  https://doi.org/10.1016/j.ejps.2026.107540
Anal Chem. 2026 May 05.

Live-Cell Derivatization Enables High-Throughput Analysis of Thiols with CyESI-MS.

Kexin Xu, Jinlei Yang, Siyuan Pan, Murong Du, Runsong Cheng, Jingya Cao, Boya Zhao, Sichun Zhang, Xinrong Zhang.

Detecting thiol metabolites at the single-cell level is crucial for unraveling their roles in redox regulation and disease pathogenesis, but their detection is hindered by weak mass spectrometry (MS) signals and severe ion suppression due to their low abundance. Herein, we develop a live-cell derivatization strategy coupled with label-free mass cytometry (CyESI-MS) for sensitive and high-throughput analysis of thiols in single cells. Tetramethylrhodamine-5-maleimide (TAMRA-5-maleimide) was employed as the derivatization reagent, which can react specifically with thiols via its maleimide group, boost MS signal response through the rhodamine moiety, and exhibit good biocompatibility. Using this strategy combined with CyESI-MS, we successfully detected five thiol metabolites (GSH, Cys, Cys-Gly, hCys, and NCys) in single cells. The reliability of this method was further validated in multiple-cell application scenarios. By expanding single-cell coverage of low-abundance metabolites, this strategy holds promise for advancing redox biology and cancer research.

DOI: https://doi.org/10.1021/acs.analchem.5c07995
Anal Chem. 2026 May 04.

Improving Isotope Ratio Accuracy in Metabolic Labeling Using Orbitrap Mass Spectrometry: A Machine Learning Correction Algorithm for Metabolic Flux Analysis.

Zhenwen Yu, Naveed Ziari, Marc K Hellerstein.

Stable isotope labeling is widely used to study metabolic fluxes. Mass spectrometry is the primary tool for measuring isotope ratios in biomolecules, but there are often trade-offs between mass resolution, sensitivity, analytic speed, and, most importantly for flux measurements, accuracy and precision of relative isotope abundances. Orbitrap mass spectrometers have been found to exhibit high isotope ratio measurement accuracy and precision in targeted measurements on a narrow m/z range but are biased in untargeted measurements of multiple biomolecules concurrently across a wide m/z range. This measurement bias is known to be caused by multiple factors, including the ion signal intensity. Here, we developed a scan-by-scan, machine-learning-based correction method to address the bias and predict bias-free mass isotopomer ratios. The fully trained random forest model reduces the mean absolute percentage error of isotopic measurements in detected metabolites for the M1 mass isotopomer from 21.3 to 3.5% and for M2 from 25.8 to 3.1%. The model also provides a ranking of factors by their impact on the measurement bias, and it was found that the ion signal intensity/TIC ratio of individual scans is the most dominant and is a previously neglected bias-causing factor. Use of this correction model improves the accuracy of metabolic flux measurements from heavy water (2H2O) labeling studies. Our correction method overcomes the limitation that current Orbitrap mass spectrometers cannot achieve isotope measurement accuracy in methods covering a wide m/z range. The approach presented here enables stable isotope labeling experiments to be high-throughput and may advance stable isotope labeling toward untargeted "fluxomics".

DOI: https://doi.org/10.1021/acs.analchem.5c06638
Rapid Commun Mass Spectrom. 2026 Aug 15. 40(15): e70097

Mass Spectrometry Based Multi-Omics Integration: Analytical Methods and Biological Applications.

Jainendra Kumar Battineni, Yaso Deepika Mamidisetti, Mounika Kuchukuntla, Divya Amaravadi, Sahithya Ravali Ravula, Rajendra Kumar Jadi.

   RATIONALE: Biological systems are regulated through strongly interconnected molecular layers that cannot be accurately resolved using single-omics approaches. Although genomics and transcriptomics provide essential regulatory information, they often face obstacles to reflect functional molecular outcomes. Mass spectrometry (MS)-based multi-omics integration is currently recognized as a central analytical strategy to overcome this limitation by enabling direct, high-resolution measurement of proteins, metabolites, and lipids, thereby supporting systems-level biological interpretation and translational discovery.
METHODS: This review critically examines mass spectrometry-based multi-omics approaches through analysis of published literature, with a focus on integrating proteomic, metabolomic, lipidomic, and spatial omics data. Computational frameworks and translational applications relevant to biomarker discovery and precision medicine are highlighted.
RESULTS: MS-centered multi-omics integration significantly enhances molecular coverage, quantitative accuracy, and pathway-level interpretation by combining various analytical layers. Applications across cancer biology, metabolic disorders, neurodegenerative diseases, and environmental research have exhibited improved biomarker robustness and mechanistic resolution contrast with single-omics studies. Recent developments in spatial and single-cell MS address cellular heterogeneity, while integrative computational approaches minimise challenges associated with data complexity, normalization, and cross platform variability.
CONCLUSIONS: Mass spectrometry-based multi-omics integration represents a rapidly evolving analytical approach for systems biology and translational research. Continued advances in MS instrumentation, acquisition strategies, and computational integration are expected to further improve biological interpretability that fastens the discovery of clinically and biologically relevant molecular signatures.

Keywords:  lipidomics; mass spectrometry; metabolomics; multi‐omics integration; proteomics; spatial MS

DOI:  https://doi.org/10.1002/rcm.70097
J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 28. pii: S1570-0232(26)00188-1. [Epub ahead of print]1278 125099

Quantification of nirmatrelvir concentrations in rat plasma, cerebrospinal fluid, and peripheral blood mononuclear cell extracts using LC-MS/MS.

Timothy M Mykris, Lee Winchester, Johid R Malik, Ukamaka O Modebelu, Courtney V Fletcher, Sean N Avedissian.

  Highly sensitive LC-MS/MS methods were developed and validated to quantify nirmatrelvir (NMR). Although the plasma pharmacokinetics (PK) of NMR have been well characterized, its distribution into other biologically relevant compartments, such as cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMCs), and tissues, remains poorly understood. Quantitative assessment of NMR across these matrices is essential for evaluating central nervous system and intracellular exposure. To address this gap, we developed and fully validated three independent LC-MS/MS assays for the quantification of NMR in rat plasma, CSF, and PBMC matrices following FDA bioanalytical method validation guidelines. Calibration ranges were 20-10,000 ng/mL for plasma, 1.00-250 ng/mL for CSF, and 0.100-5.00 ng/mL for PBMCs, using NMR-D9 as the internal standard. Matrix-specific extraction procedures were optimized to address physicochemical and protein-binding differences, including a methanolic ammonium hydroxide treatment to mitigate adsorption losses in CSF. Chromatographic separation was achieved on a C18 column with a 60:40:0.1 (v/v/v) acetonitrile:water:formic acid mobile phase, and detection was performed by mass spectrometry in positive multiple-reaction-monitoring mode. All assays demonstrated excellent linearity (r2 > 0.99), precision and accuracy within acceptance criteria (< 15% CV and deviation, < 20% at the LLOQ), with no significant matrix interference or ion suppression. Stability testing confirmed consistent performance under short-term, freeze-thaw, and long-term conditions. These validated assays provide sensitive, selective, and reproducible quantification of NMR across multiple matrices and will facilitate preclinical pharmacokinetic studies aimed at understanding its distribution in sanctuary compartments relevant to SARS-CoV-2 infection.

Keywords:  Cerebrospinal fluid; LC-MS/MS; Nirmatrelvir; PMBCs; Tissues

DOI:  https://doi.org/10.1016/j.jchromb.2026.125099
J Am Soc Mass Spectrom. 2026 May 06.

From Mythology to Methodology: Untargeted Single-Cell Metabolomics in R with MeDUSA.

Laura Ann Castaneda, Eric Hetzel, Fercan Gül, Thomas Hankemeier, Ahmed Ali.

  Direct infusion-based single-cell metabolomics analysis has the potential to isolate the causes of drug resistance and cancer progression; however, processing and analyzing the data generated remains a challenge. While many packages exist for metabolomics analysis, they are not optimized for direct infusion-based single-cell measurements, which do not rely on chromatographic separation and are typically noisier than traditional population-level methods. To address this gap, the MeDUSA (Metabolomics of direct-infusion untargeted single-cell analysis) R package was developed. MeDUSA was built especially for direct infusion-based single-cell metabolomics, with modularity, noise filtering, and user-customization in mind. In this work, we introduce the package, how to use it, and implement it in a single-cell metabolomics experiment to identify the differences between two cell lines. MeDUSA compromises several functions that deal with file import, peak picking, spectral processing, statistical analysis, and feature annotation. Each function is defined with the purpose, usage, parameters, default values, and output of an example data set. MeDUSA was built to be a modular platform that aims to be a foundation to be built upon with additional modules for the single-cell metabolomics field.

Keywords:  data analysis; direct infusion; metabolomics; single cell

DOI:  https://doi.org/10.1021/jasms.5c00297
Anal Chem. 2026 May 08.

A Unified Bayesian Framework for Cross-Technology Collision Cross Section Postcalibration Correction and Multilaboratory Harmonization.

Yi-Hui Zhou, George Sun.

Reliable comparison of collision cross section (CCS) measurements across ion mobility platforms remains a key challenge for standardized molecular identification. We present a hierarchical, data-driven postcalibration correction framework that harmonizes CCS values across drift tube (DTIMS), trapped ion mobility (TIMS), and traveling wave (TWIMS) instruments, enabling quantitative cross-technology comparison despite differing calibration methods. Using a multilaboratory dataset of 840 measurements for 347 compounds, the framework reduced intertechnology CCS variability by approximately 95%, bringing residual differences within instrumental precision limits. Leave-one-compound-out validation demonstrated robust cross-technology transfer, lowering the median absolute percentage error from 8.9% to 3.2% and achieving 94.6% empirical coverage of 95% prediction intervals. The approach requires as few as three reference compounds, reducing recalibration workload by more than half, and provides full uncertainty estimates for probability-based database matching. This statistically grounded yet practical strategy enables harmonized, uncertainty-aware CCS databases and supports reproducible compound identification across laboratories and ion mobility technologies.

DOI: https://doi.org/10.1021/acs.analchem.5c06667
J Am Soc Mass Spectrom. 2026 May 06.

Multi-Laboratory Assessment Reveals Variable Ion Species Profiles in Electrospray Ionization Mass Spectrometry.

Melanie T Odenkirk, Jacqueline M Chaparro, Nathan T Montgomery, Katrina L Leaptrot, Stacy D Sherrod, Jody C May, Henriëtte van Eekelen, Bert Schipper, Juliana Chaura, Gabriel Esteban Velez, Wimonphan Chathiran, Cole R Michel, Katrina A Doenges, Richard Reisdorph, Mari Maeda-Yamamoto, Luis Valdiviez, Uri Keshet, Jeremiah Wells, Scott Mackell, David Bell, Sebastian Tapia Leiva, Simon Partridge, Daniela Rago, Linda Ahonen, Yuki Ito, Margaret L Read, John C Evans, Macy J Gruszczynski, Leokadja Szalach, Stephen C Boyko, Steven Watkins, Rodrigo Ledesma Amaro, Oliver Fiehn, Nichole Reisdorph, Warangkana Srichamnong, Andres Jaramillo-Botero, Ric C H de Vos, Robert D Hall, John A McLean, Corey D Broeckling, Jessica E Prenni.

  Electrospray ionization (ESI) is a foundational technique in mass spectrometry (MS) widely applied to measure nonvolatile molecules in diverse chemical and biological samples. Yet variation in ESI-MS instrumentation, operating settings, and parameters such as solvent composition creates specific, local ionization environments that drive the formation of select ion species for individual analytes. As ESI continues to advance analytical discovery, understanding the extent to which variation in ion species formation impacts intra- and inter-experimental results is essential. Here, we assessed ion species formation by analyzing an internal retention time standard (IRTS) mixture across ten laboratories employing high-resolution ESI-MS instrumentation from four vendors (Agilent Technologies, ThermoFisher Scientific, Shimadzu Corporation, and Waters Corporation). Instrument vendors were considered not as a benchmark of performance, but as a practical framework to capture differences in source design, ion optics, and analyzer/detectors that are inherently coupled to commercial platforms. Despite the use of standardized extraction and chromatographic protocols, differences in instrument configuration, source conditions, and method execution resulted in variation in ion species formation across vendors, among laboratories using instruments from the same vendor, and even within individual laboratories. These findings demonstrate that, even with standardized methods, the collective influence of local ionization environments on ion species formation remains a critical obstacle for interpreting LC-MS small molecule data and improving reproducibility and comparability across studies.

Keywords:  electrospray (ESI); ionization; mass spectrometry (MS); metabolomics; molecule

DOI:  https://doi.org/10.1021/jasms.6c00011
Food Res Int. 2026 Jul 01. pii: S0963-9969(26)00849-5. [Epub ahead of print]235 119172

Development and validation of a new derivatization-based quantitative analysis of multi-components by single marker strategy for simultaneous quantification of biogenic amines in fermented wine.

Yue-Cai Hou, Xiao-Feng Huang, Ying Xue, Zhi-Xiang Yang, Lin-Sen Qing.

  Simultaneous quantitative analysis of multiple biogenic amines (BAs) in food is challenging due to the high cost of reference standards, weak ultraviolet absorption, and complex matrix interference. To address these limitations, this study developed a strategy integrating dansyl chloride (DNS-Cl) derivatization with the quantitative analysis of multi-components by single marker (QAMS) approach to enable simultaneous quantification of nine BAs in fermented wine using HPLC-UV. A key methodological innovation of this work is the first-time adoption of a weighted scoring system for the systematic selection of the optimal internal standard (IS) for QAMS. This system comprehensively evaluates the stability of the relative correction factor (RCF), retention time suitability, and response intensity, moving beyond traditional empirical choices. Through this rigorous evaluation, tyramine (Tyr) was selected as the optimal internal standard. The established RCFs proved reproducible and stable across different instruments and operational conditions (RSD < 10%). Method validation demonstrated good linearity (R2 > 0.9985), precision (RSD < 2.28%), and accuracy (recovery: 93.92%-107.43%). Bland-Altman analysis showed that 100% of data points lay within the 95% confidence interval, demonstrating strong agreement with conventional external standard methods. In conclusion, the developed derivatization-based QAMS method is accurate, reliable, and cost-effective for the simultaneous quantification of nine biogenic amines in fermented wine, offering a practical alternative to traditional approaches. This method provides a low-cost analytical tool for quality control of BAs in fermented foods and expands the applicability of QAMS to compounds with weak UV absorption.

Keywords:  Biogenic amines; Dansyl chloride derivatization; HPLC-UV; Quantitative analysis of multi-components by single marker

DOI:  https://doi.org/10.1016/j.foodres.2026.119172
J Chromatogr A. 2026 May 02. pii: S0021-9673(26)00393-6. [Epub ahead of print]1780 467064

Analysis of weight-loss compounds in health supplements by ultrasonic extraction, dispersive sorbent cleanup, and liquid chromatography tandem mass spectrometry quantification.

Khanh Hung Nguyen, Anh Quoc Hoang, Thi Trang Vu, Anh Phuong Vu, Viet Ngan Le, Quang Hung Nguyen, Cong Khanh Cao, Viet Chien Dinh, Thi Oanh Nguyen, Quang Huy Nguyen, Thi Anh Huong Nguyen, Thi Hong Hao Le.

  Current society is facing the global surge in obesity rates, making people more aware of the importance of body weight control and increasing the consumption of weight-loss supplements due to their over-the-counter status. However, certain producers have intentionally adulterated supplements with prescription weight-loss drugs without label declaration, necessitating suitable analytical methods for routine analysis of illegal weight-loss compounds in health supplements. In this study, a rapid, effective, and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 11 wt-loss compounds in three supplement forms (i.e., hard capsule, soft capsule, and tea powder). A suitable analytical procedure was selected via the optimization of LC-MS/MS parameters and sample preparation conditions. The samples were ultrasonicated with methanol, and the extracts were cleaned up using dispersive solid-phase extraction with C18 and carbon sorbent. The method was validated for specificity, linearity, method detection and quantification limits, precision, and accuracy, producing good satisfaction. The method was applied to analyze 82 health supplement samples collected from the Vietnamese market, in which seven samples were found to be adulterated with at least one of the target compounds. Sibutramine, phenolphthalein, and benzylsibutramine were detected in 7/82, 5/82, and 2/82 samples with concentration ranges of 0.44-16.4, 0.54-2.14, and 0.14-1.43 mg/g, respectively. These results indicated the method applicability to the quality control of health supplements in developed and emerging countries.

Keywords:  Adulteration; Health supplements; LC-MS/MS; Weight-loss compounds

DOI:  https://doi.org/10.1016/j.chroma.2026.467064
Crit Rev Anal Chem. 2026 May 08. 1-29

Critical Evaluation of Analytical Strategies for the Quantification of Procaine and Its Metabolites Across Diverse Matrices.

Hemn A H Barzani, Rebaz Anwar Omer, Nergz Bayiz Abdulrahman, Hunar Yasin Muhammad, Seerwan Hamadameen Sulaiman.

  Procaine (PC) is an ester-type local anesthetic whose rapid enzymatic and chemical hydrolysis to p-aminobenzoic acid (PABA) makes intact-drug quantification exceptionally difficult across plasma, serum, urine, pharmaceutical formulations, and environmental waters, where PC exists only transiently and at ultra-trace levels; this review therefore aims to critically compare chromatographic, electrochemical, and spectroscopic strategies for the selective and sensitive measurement of PC and its metabolites. The evidence shows that physical separation by HPLC is indispensable for resolving PC from PABA and excipients, with fluorescence-enhanced HPLC achieving ng/mL sensitivity, while LC-MS/MS offers demonstrated analytical feasibility molecular selectivity and sub-ng to pg/mL limits enabling simultaneous PC-PABA profiling in ≤5 min; nanostructured and molecularly imprinted electrochemical sensors exploit the electroactive para-aminobenzoate moiety to provide rapid, matrix-tolerant nanomolar to sub-nanomolar detection that may reduce the extent of ex vivo hydrolysis due to rapid analysis and minimal sample handling time. Spectroscopic assays are suitable for high-throughput pharmaceutical and environmental screening, but their applicability is limited by drug instability and cross-reactivity. Given the relative scarcity of chromatographic studies on PC despite its instability-driven analytical complexity, future work should prioritize stabilization-coupled LC-MS/MS and next-generation electrochemical platforms, together with green, miniaturized workflows and AI-assisted optimization, to enable robust, high-confidence quantification of PC in real-world matrices.

Keywords:  Electrochemical analysis; LC–MS/MS; chromatography; procaine; spectroscopy

DOI:  https://doi.org/10.1080/10408347.2026.2665383
BMC Chem. 2026 May 08.

A novel green QbD-optimized RP-HPLC bioanalytical method for the simultaneous determination of doxorubicin and rifampicin in human plasma.

Galal Magdy, Shaimaa Ebrahim, Fathalla Belal, Ahmed M Abdel-Megied.

  Doxorubicin hydrochloride (DOX) remains a cornerstone in the treatment of numerous malignancies. Recent research has focused on improving the efficacy of chemotherapeutic regimens through synergistic combinations. Rifampicin (RFP), in addition to its well-known antimicrobial properties, exhibits chemosensitizing potential, making it a promising adjuvant to DOX therapy. In this study, a robust and eco-friendly HPLC method was developed and optimized for the simultaneous determination of DOX and RFP. Chromatographic separation was achieved on a C18 column (150 × 4.6 mm, 5 μm particle size) at 25 °C, using an isocratic mobile phase composed of acetonitrile and phosphate buffer (0.02 M, pH 5.43) in a 34.85:65.15 (v/v) ratio, with a flow rate of 0.8 mL/min and UV detection at 254 nm. The method was systematically optimized using a Quality-by-Design (QbD) approach with a full factorial design to assess the influence of key variables on chromatographic performance. The optimized chromatographic separation was achieved within 7 min, with retention times (Rts) of 3.26 min of DOX and 6.62 min for RFP. Excellent linearity was obtained over the ranges of 1.0-40.0 µg/mL for DOX and 1.0-30.0 µg/mL for RFP, with high determination coefficients (r² ≥ 0.999) and lower detection limits of 0.44 µg/mL and 0.39 µg/mL and quantitation limits of 1.35 µg/mL and 1.18 µg/mL, respectively. Greenness evaluation using the Analytical Greenness (AGREE) and Analytical Green Star Area (AGSA) metrics confirmed the method's high environmental sustainability. When applied to spiked human plasma samples, recovery values ranged from 92.38% to 107.20% with low %RSD, demonstrating strong accuracy, precision, and bioanalytical applicability. Overall, the developed method represents the first simple, sensitive, and environmentally benign analytical approach for the simultaneous quantification of doxorubicin HCl and rifampicin in biological matrices, making it highly suitable for routine therapeutic drug monitoring (TDM) in clinical and pharmacokinetic studies.

Keywords:  Doxorubicin HCl; Greenness; Liquid chromatography; Plasma; Rifampicin; Therapeutic drug monitoring

DOI:  https://doi.org/10.1186/s13065-026-01809-x
J Sep Sci. 2026 May;49(5): e70411

From Columns to Clouds: Emerging Interfaces and Ion Sources for Coupling Analytical Separations With Mass Spectrometry.

Chamarthi Maheswar Raju, Pawel L Urban.

Although mass spectrometry (MS) is a powerful analytical technique, it is vulnerable to matrix interference, and it does not distinguish ions with the same m/z (isobaric interference). Therefore, for the past few decades, numerous efforts have been made to enable coupling separation systems with MS. Apart from a set of mainstream hyphenation approaches (such as those using inductively coupled plasma ionization, electron ionization, and electrospray ionization), a number of less conventional ways to couple separation systems with MS have been devised. Extraordinary methods for coupling gas chromatography, liquid chromatography, capillary electrophoresis, thin-layer chromatography, supercritical fluid chromatography, and other unconventional interfaces and ion sources have been disclosed in the literature. Some of these techniques extend the analyte coverage of traditional hyphenated techniques by enabling the detection of less polar compounds. Others speed up analysis or decrease the cost. Another notable trend is the implementation of the so-called ambient ionization techniques for coupling separations with MS.

DOI: https://doi.org/10.1002/jssc.70411
Se Pu. 2026 May 08. 44(5): 547-554

[Determination of perfluorinated compounds， antibiotics and pesticides in drinking water by automated solid phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry].

Jing Liang, Jiali Feng, Dong Zeng, Liping Zhou, Xuan Zhong, Yi Li, Dongyang Chen.

  Emerging organic contaminants in drinking water, characterized by biological toxicity and environmental persistence. Developing effective strategies for monitoring and controlling these contaminants in drinking water has become an urgent need to ensure drinking water safety. However， the current detection technologies for emerging organic contaminants in drinking water mainly focus on analyzing a single type of pollutant， which increases in detection costs and fails to meet the urgent demand for rapid， multi-analyte screening in water quality monitoring. This study established a novel analytical method， which was based on the automatic solid phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry method （SPE-UPLC-MS/MS）， enabling the simultaneous detection of 30 contaminants with diverse physicochemical properties in drinking water. The 30 contaminants included perfluorinated compounds （PFCs）， antibiotics and pesticides. The water samples were pretreated automatically by an automatic solid phase extraction instrument， and then purified and enriched through an OASIS HLB solid phase extraction column. The 30 contaminants were separated on an ACQUITY UPLC HSS T3 column （100 mm×2.1 mm， 1.7 μm） with a gradient mobile phase consisting of 5 mmol/L ammonium acetate （containing 0.1% formic acid） and acetonitrile. The samples were analyzed and determined by the positive/negative switching mode of the electrospray ionization source， and quantified by the external standard method. The results showed that the 30 compounds had good linear relationships within the mass concentration ranges of 0.10-200.0 μg/L， and the correlation coefficients （r） greater than 0.990. The limits of detection were 0.01-1.0 ng/L， and the limits of quantification were 0.03-3.0 ng/L. The 30 compounds exhibited recoveries in the range of 70.2%-120.0% at three levels （0.20， 1.0， 10.0 μg/L）， and the relative standard deviations （RSDs， n=6） were 3.2%-9.6%. Water samples from nine counties and districts in Hunan Province were analyzed using the developed method. The 16 compounds were detected， with the mass concentration range of 0.1-9.9 ng/L. Among these， atrazine was the most frequently detected， whereas the highest concentration observed was chlorantraniliprole. According to the control requirements specified in GB 5749-2022 Standards for Drinking Water Quality， the contents of perfluorooctanoic acid， 2，4-dichlorophenoxyacetic acid， atrazine， carbofuran， and bentazone were significantly lower than the national standard limits， indicating a low health risk associated with these compounds in the source water of the studied regions. This method is efficient， rapid， and cost-effective， which is suitable for the simultaneous determination of PFCs， antibiotics， and pesticides in drinking water. This method provides strong technical support for the control and treatment of new contaminants in drinking water.

Keywords:  antibiotics; perfluorinated compounds （PFCs）; pesticides; solid phase extraction （SPE）; ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）; water

DOI:  https://doi.org/10.3724/SP.J.1123.2025.08023
Se Pu. 2026 May 08. 44(5): 565-574

[Determination of six whitening ingredients in cosmetics by ultra performance liquid chromatography coupled with photodiode array detector and corona charged aerosol detector].

Xiaofang Li, Ye Wang, Fan Fan, Wei Zhou, Bin Chen, Huajin Shi, Guoqiang Cai, Ying Liu, Yibo He.

  Whitening is a core function of cosmetics， and ingredients including acetyl glucosamine， tranexamic acid， nicotinamide， phenethyl resorcinol， glabridin， and ascorbyl tetraisopalmitate are widely used due to their clearly established whitening mechanisms. However， existing analytical methods face notable limitations： photodiode array detectors （PDA） cannot reliably detect weakly or non-ultraviolet （UV）-absorbing components； evaporative light scattering detectors （ELSD） suffer from inadequate sensitivity for low-concentration analytes； mass spectrometry （MS） is costly； and single-column cannot achieve the separation of ingredients with significantly different polarities. To address these challenges， this study established an analytical method for the determination of the six whitening ingredients in cosmetics using ultra performance liquid chromatography coupled with a photodiode array detector and a corona charged aerosol detector （UPLC-PDA-CAD）. For sample pretreatment： 0.2 g of sample was weighed， and 10 mL dichloromethane and 10 mL deionized water were added for vortex extraction of whitening ingredients. The mixture was then centrifuged for phase separation. The aqueous layer and dichloromethane layer were collected separately. The aqueous layer was washed twice with dichloromethane； all dichloromethane fractions were combined， concentrated to less than 1 mL under a nitrogen stream， and finally made up to volume with isopropanol. Both the aqueous layer and the reconstituted isopropanol solution were filtered through 0.22 μm hydrophilic polytetrafluoroethylene （PTFE） membranes before UPLC injection. For chromatographic separation， a Waters HSS T3 column （150 mm×2.1 mm， 1.7 μm） was selected. The column temperature was set at 40 ℃， the injection volume was 1 μL， and the flow rate was 0.3 mL/min. The mobile phase consisted of isopropanol， acetonitrile， and 20 mmol/L ammonium acetate solution （pH adjusted to 4.5 using formic acid）， using a gradient elution program. For detection， a tandem PDA-CAD system was used： PDA （scan range 200-400 nm） was employed for components with UV absorption， while CAD （nebulization temperature 35 °C， acquisition frequency 5 Hz） was used for components with weak UV absorption. Quantification was performed using the external standard method.Method validation results showed good linear relationships for all six whitening ingredients within their respective concentration ranges. The correlation coefficients （r） were all greater than 0.999. The limits of detection （LODs， S/N=3） were 5.0-50.0 µg/g， and the limits of quantification （LOQs， S/N=10） were 12.0-120.0 µg/g. Spiked recovery tests were conducted on negative cosmetic matrices （emulsion， cream， oil） at low， medium， and high levels. The recoveries ranged from 92.8% to 110.1%， and the relative standard deviations （RSDs， n=6） were 0.12%-5.45%， indicating excellent precision and accuracy. This method was applied to seven commercially cosmetics. The results revealed that all target whitening ingredients declared on the product labels were detected， with significant differences in their content. Nicotinamide was the most frequently detected compound， found in five products， and also exhibited the highest concentrations， ranging from 0.19% to 2.29%. Phenethyl resorcinol was detected in three products， with contents ranging from 0.02% to 0.52%. Ascorbyl tetraisopalmitate was detected in two products， at 0.09% and 3.08%， respectively. Acetyl glucosamine， tranexamic acid， and glabridin were each detected in only one product. In conclusion， this established UPLC-PDA-CAD method is simple， efficient， sensitive and accurate. It effectively overcomes the technical challenge of detecting whitening ingredients with widely varying polarities， offering reliable technical support for cosmetic quality control， regulatory supervision， and evaluation of product whitening efficacy and potential sensitization risks.

Keywords:  corona charged aerosol detector （CAD）; cosmetics; photodiode array detector（PDA）; ultra performance liquid chromatography（UPLC）; whitening ingredients

DOI:  https://doi.org/10.3724/SP.J.1123.2025.09016
Biomed Chromatogr. 2026 Jun;40(6): e70483

Comprehensive Impurity and Degradation Profiling of Iptacopan Using a White-Green Stability-Indicating HPLC Method Coupled With LC-MS/MS and In Silico Toxicity Evaluation.

Satyanarayana Reddy Jaggavarapu, Ajmal R Bhat, Venkateswara Rao Anna.

  A comprehensive, stability-indicating, and environmentally sustainable analytical strategy was developed for the impurity and degradation profiling of iptacopan. Optimum separation was achieved on a Hypersil BDS C18 (250 × 4.6 mm, 5 μm) column using 0.8-mL/min isocratic flow of 10-mM ammonium acetate buffer (pH 4.5) and ethanol (60:40, v/v) with 234-nm UV detection. The method produced excellent linearity (r2 ≥ 0.9995), precision (%RSD ≤ 0.75), accuracy (98.32%-100.26%), and robustness. Forced degradation studies generated four distinct degradation products (DPs) under acidic and oxidative stress, characterized by LC-MS/MS. DP 3 showed a +30-Da mass increase (m/z 453.4996), consistent with an indole-carboxylic acid analogue, whereas DP 1 (m/z 439.4730) exhibited a -14-Da shift, indicating demethylation. DP 2 displayed a -42-Da difference (m/z 381.4369) due to dealkylation, and DP 4 showed a +14-Da shift (m/z 437.5002), corresponding to a linker ketone analogue. In silico toxicity assessment suggested moderate toxicity for most DPs, with DP 4 showing a comparatively favorable profile. Greenness and whiteness evaluation using AGREE (0.82), AGREE-Prep (0.81), GAPI (E-factor = 10), and RGB-12 (78.0%) confirmed excellent environmental and practical performance. The method provides a robust and sustainable platform for quality control and stability assessment of iptacopan.

Keywords:  degradation products; iptacopan; method greenness; method whiteness; process‐related impurities

DOI:  https://doi.org/10.1002/bmc.70483
Talanta. 2026 Apr 24. pii: S0039-9140(26)00550-3. [Epub ahead of print]307 129894

Feasibility verification of fingerstick capillary microsampling replacing venipuncture for therapeutic drug monitoring of 12 antibiotics using a rapid LC-MS/MS assay.

Feng Chen, Ziwei Deng, Yueping Jiang, Yayun Tan, Xiaoxia Yang, Huanhuan Li, Wendan Zhang, Xiangjin Shu, Aimin Wang, Chengfeng Qiu.

  Therapeutic drug monitoring (TDM) is essential for optimizing antimicrobial therapy and curbing resistance, yet conventional venipuncture limits its widespread application due to invasiveness and operational complexity. This study aimed to establish a rapid LC-MS/MS method for simultaneous quantification of 12 antimicrobial agents and to evaluate the feasibility of using fingertip capillary serum as an alternative to venous serum for TDM. A liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous quantification of vancomycin, linezolid, voriconazole, sulfamethoxazole, trimethoprim, piperacillin, cefoperazone, ceftazidime, ceftobiprole, meropenem, imipenem, and cilastatin in human serum. The method was applied to analyze 379 paired clinical samples (fingertip serum versus venous serum), and agreement between the two sampling approaches was assessed using Deming regression and Bland Altman analysis. The method required only 10 μL of serum and a total run time of 4 min. Deming regression slopes ranged from 0.910 to 1.017. Bland Altman analysis showed that 100.0% of the data points fell within ±20.0% limits of agreement for all antibiotics except vancomycin (96.7%) and cilastatin (97.5%). This study provides large scale clinical evidence demonstrating high concordance between fingertip serum and venous serum concentrations for 12 antimicrobial agents. By directly obtaining serum, this approach fundamentally avoids the hematocrit effect and matrix conversion issues inherent in whole blood microsampling, offering a minimally invasive TDM strategy fully aligned with the gold standard matrix. This strategy is particularly suitable for special populations such as children, the elderly, and critically ill patients.

Keywords:  Antimicrobial agents; Capillary microsampling; Fingertip blood; LC-MS/MS; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.talanta.2026.129894