bims-metlip 2025-09-21 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–09–21
27 papers selected by
Sofia Costa, Matterworks

Quantitative profiling of lipid mediators in sperm cells through on-line dilution on-line polymer matrix-based solid-phase extraction liquid chromatography with mass spectrometric detection.
A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quick and efficient quantification of five fat-soluble vitamins.
Current trends in sample preparation for liquid chromatography - mass spectrometry analysis of catecholamines and their metabolites in biological samples.
Reliable in-house LC-MS/MS method for steroid hormone analysis: Validations and comparisons.
A reliable LC-MS/MS method for urinary oxalate determination and its comparison with analogous assays.
Multiomics Analysis of Dried Plasma Spots Using Stable Isotope Internal Standards for Biomarker Discovery.
Quantitative UHPLC-MS/MS Analysis of Gemcitabine and Its Active and Inactive Metabolites in Pig Plasma and Lung Tissue in Preparation of Locoregional Therapies for Non-Small Cell Lung Cancer.
Features of sample preparation of cell culture samples for metabolomic screening by LC-MS/MS.
MSI.EAGLE: An Open-Source GUI for Streamlined Mass Spectrometry Imaging Analysis.
Two-Dimensional LC × DMS Analysis of 34 PFAS Compounds.
MassVision: An Open-Source End-to-End Platform for AI-Driven Mass Spectrometry Imaging Analysis.
MSnLib: efficient generation of open multi-stage fragmentation mass spectral libraries.
Comprehensive Metabolic Profiling of Resigratinib, a Novel FGFR Inhibitor, Using Integrated LC-MS/MS and LC-Orbitrap-HRMS.
Efficient and Wide Chemical-Space Ionization of Organic Contaminants Using LC-MS with a Miniaturized Plasma Source Applying Different Discharge Gases.
Toward standardized epitranscriptome analytics: an inter-laboratory comparison of mass spectrometric detection and quantification of modified ribonucleosides in human RNA.
Reliable LC-MS/MS method development and validation for the determination of methylmalonic acid, methylcitric acid, malonic acid, ethylmalonic acid, and total homocysteine in dried blood spots.
First Specific Analytical Method for Quantification of Halide Ions by UHPLC-MS Using Reverse Phase Chromatography C18.
Single-cell metabolite annotation by tandem mass spectrometry imaging and ab initio molecular dynamics-based fragmentation.
Development and validation of general urine screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high resolution mass spectrometry (LC-HRMS).
A complementary multi-aptamer approach for efficient extraction of steroid hormones and application to their determination in biological samples using Q-TOF LC-MS.
Validation and quantitation of six neurotransmitters in rat plasma and brain using liquid chromatography quadrupole-time-of-flight mass spectrometry.
A Novel Chemical-Space-Dependent Strategy for Compound Selection in Non-target LC-HRMS Method Development Using Physicochemical and Structural Data.
Inner-wall biochar-coated pipette tip microextraction for rapid and sustainable determination of tyrosine kinase inhibitors in plasma.
A UPLC-MS/MS method for the determination of 2-nitro-1,3-propanediol, a metabolite of Bronopol, in human urine.
A thorough LC-MS/MS strategy to quantify abaloparatide in rat plasma: method development, validation and application.
Eco-friendly microextraction technique using SFO-DLLME coupled with HPTLC for plasma analysis of metformin and imeglimin.
Advances in the chemical analysis of nitrite in environmental and biological samples.

Anal Methods. 2025 Sep 15.

Quantitative profiling of lipid mediators in sperm cells through on-line dilution on-line polymer matrix-based solid-phase extraction liquid chromatography with mass spectrometric detection.

Jörg Fabian, Matthias Lehr.

An automated on-line dilution on-line solid-phase extraction liquid chromatography-mass spectrometry method was developed for the straightforward and versatile quantitative profiling of lipid mediators such as various eicosanoids, endocannabinoids, and arachidonic acid in human sperm. The LC-MS/MS system utilized a polymer matrix-based trap column (TurboFlow Cyclone™) for sample pre-treatment, followed by chromatographic separation using an ODS 3 μm, 100 × 2 mm column, and detection via a triple-quadrupole mass spectrometer equipped with an electrospray interface. The sample preparation process employed a simple "dilute and shoot" approach, with a centrifugation step to remove proteins. This method has significantly improved sensitivity and selectivity while minimising matrix effects, with limits of quantification in the low picogram range for most analytes. Linearity, accuracy, precision, and recovery all met the required criteria for bioanalytical method validation. This LC-MS/MS approach was successfully applied to determine basal levels of selected eicosanoids, endocannabinoids, and arachidonic acid in human spermatozoa.

DOI: https://doi.org/10.1039/d5ay00250h
Clin Chim Acta. 2025 Sep 16. pii: S0009-8981(25)00494-2. [Epub ahead of print] 120615

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quick and efficient quantification of five fat-soluble vitamins.

Si-Wei Li, Yi Luo, Wei Chen, Chao Huang, Yan Chen, Xiao-Yue Hong.

   BACKGROUND: Deficiencies in fat-soluble vitamins (FSVs) have been linked to various systemic disorders. In this study, we introduce a novel and straightforward assay for the analysis of FSVs, suitable for large-scale sample screening while reducing laboratory testing costs.
METHODS: The method, termed sodium-acetonitrile extraction (SoAce), was developed and validated for the quantification of selected FSVs, including Vitamin A (retinol), Vitamin D2 (25-hydroxyvitamin D2), Vitamin D3 (25-hydroxyvitamin D3), Vitamin E (α-tocopherol), and Vitamin K1 (phylloquinone). A saturated sodium chloride solution combined with acetonitrile was employed as the extraction reagent. Mass spectrometric detection was carried out using a triple quadrupole mass spectrometer in positive ion mode via an APCI source. Method validation was conducted in accordance with Chinese regulatory guidelines and the C62-A standard.
RESULTS: Using the SoAce-liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, FSVs were successfully separated and quantified within 30 min. The assay demonstrated excellent linearity, with all regression coefficients exceeding 0.996. The lower limit of quantification (LLOQ) was determined as 12.5 ng/mL for Vitamin A, 0.31 ng/mL for Vitamin D2, 1.56 ng/mL for Vitamin D3, 625 ng/mL for Vitamin E, and 0.08 ng/mL for Vitamin K1. Specificity, stability, accuracy, and precision were all evaluated and met the required validation criteria. A comparative analysis against an existing commercial FSV kit using the same instrumentation revealed strong correlation, with coefficients ranging from 0.928 to 0.962.
CONCLUSIONS: The developed SoAce-LC-MS/MS method fulfills the requirements for rapid and reliable assessment of FSV status in human serum. Further studies are warranted to fully explore the potential applications and clinical utility of this assay.

Keywords:  Fat-soluble vitamins; LC-MS/MS; Pretreatment; Sodium-acetonitrile method

DOI:  https://doi.org/10.1016/j.cca.2025.120615
Talanta. 2025 Sep 07. pii: S0039-9140(25)01328-1. [Epub ahead of print]298(Pt A): 128837

Current trends in sample preparation for liquid chromatography - mass spectrometry analysis of catecholamines and their metabolites in biological samples.

Maria Khalikova, Klára Faistauerová, Miroslav Lísa.

  Catecholamines, including dopamine, norepinephrine, and epinephrine, are essential neurotransmitters and hormones that regulate a wide spectrum of physiological functions, such as stress response, mood, and cardiovascular activity. They are closely associated with various health conditions, making both them and their metabolites of great interest as potential biomarkers for diagnosing and monitoring the treatment of different human diseases. The use of catecholamines and their metabolites in research and clinical practice requires sensitive and accurate determination in various biological matrices, each of which presents unique challenges. In this review, we summarize sample preparation methods for liquid chromatography-mass spectrometry (LC-MS) determination of catecholamines and their metabolites in the most widely studied biological samples. We compare methods from the literature published between 2000 and the present for LC-MS analysis of urine, blood, brain, cerebrospinal fluid, and microdialysate samples. The methods are discussed concerning the specific properties of individual sample types, with special emphasis on method validation. Future trends in sample preparation procedures are outlined, including mainly microextraction techniques and automation of sample preparation procedures.

Keywords:  Catecholamines; Liquid chromatography; Mass spectrometry; Metabolomics; Neurotransmitters; Sample preparation

DOI:  https://doi.org/10.1016/j.talanta.2025.128837
Clin Chim Acta. 2025 Sep 12. pii: S0009-8981(25)00489-9. [Epub ahead of print] 120610

Reliable in-house LC-MS/MS method for steroid hormone analysis: Validations and comparisons.

Yifan He, Mei Luo, Jinbo Hu, Qifu Li, Shumin Yang, Qianna Zhen, Li Ma.

   BACKGROUND: Accurate quantification of steroid hormones is crucial for diagnosing and monitoring endocrine disorders. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers superior specificity and sensitivity for simultaneous analysis of multiple steroids.
METHODS: We developed a high-throughput LC-MS/MS method to quantify 19 steroids in plasma and serum in a single analytical run. This method utilized optimized protein precipitation combined with solid-phase extraction for sample preparation and was rigorously validated following established guidelines. We then compared it with an IVD-CE-certified routine chemiluminescence immunoassay for 5 steroids and a commercial LC-MS/MS method for 17 steroids, using a total of 208 authentic and pooled plasma samples.
RESULTS: The developed method demonstrated strong linearity (R2 > 0.992), high sensitivity (LOD: 0.05-0.5 ng/mL), and excellent precision and accuracy (%CV < 15 %, recovery: 91.8 %-110.7 %). It correlated well with immunoassay overall (intraclass correlation coefficients, ICCs > 0.90), but showed improved accuracy particularly at lower concentrations especially for testosterone and progesterone. High concordance was observed between the two LC-MS/MS methods (ICCs > 0.96) with minimal proportional and constant biases.
CONCLUSIONS: The developed LC-MS/MS method is reliable for clinical laboratory use and offers a more accurate alternative to immunoassays. Its strong concordance with the commercially validated LC-MS/MS method demonstrates robustness and supports its clinical applicability.

Keywords:  Chemiluminescence immunoassay; LC-MS/MS; Method comparison; Steroid hormones

DOI:  https://doi.org/10.1016/j.cca.2025.120610
Clin Chim Acta. 2025 Sep 12. pii: S0009-8981(25)00488-7. [Epub ahead of print]579 120609

A reliable LC-MS/MS method for urinary oxalate determination and its comparison with analogous assays.

Ying Shen, Xia Luo, Qing Guan, Liming Cheng.

   BACKGROUND: The quantification of urinary oxalate is essential for the diagnosis of primary hyperoxaluria. This study sought to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary oxalate determination and to evaluate its concordance with an established in-house high-performance liquid chromatography (HPLC) method and a commercial LC-MS/MS assay.
METHODS: The newly developed LC-MS/MS method invloved the quantification of urinary oxalate through a straightforward single-step derivatization process using o-phenylenediamine at 120 °C for 20 min. A total of 291 urine samples were analyzed using both the in-house LC-MS/MS and HPLC methods, while 94 urine samples were evaluated using both our in-house LC-MS/MS method and a commercial LC-MS/MS assay. Additionally, a reference interval for 24-hour urinary oxalate in Chinese adults was established.
RESULTS: Oxalate exhibited strong analytical retention in reverse-phase liquid chromatography. The newly developed LC-MS/MS method demonstrated excellent linearity with a correlation coefficient exceeding 0.995, and acheived recovery rates ranging from 92.80 % to 98.73 %. The intra-assay and inter-assay coefficients of variation (CVs) were between 0.79 % to 7.46 %, with a total CV ranging from 2.07 % to 7.50 %. No significant matrix effect was observed. However, discrepancies were identified between the in-house LC-MS/MS method and both the commercial LC-MS/MS assay and the HPLC method. The reference interval was established at 0.12-1.04 mmol/24 h (10.8-93.6 mg/24 h).
CONCLUSIONS: The developed LC-MS/MS method is robust and simple, providing a reliable tool for the clinical assessment of urinary oxalate. An accurate method is essential for screening analyses in primary hyperoxaluria.

Keywords:  24-hour urine; Derivatization; Hyperoxaluria; LC-MS/MS; Oxalate

DOI:  https://doi.org/10.1016/j.cca.2025.120609
Anal Chem. 2025 Sep 17.

Multiomics Analysis of Dried Plasma Spots Using Stable Isotope Internal Standards for Biomarker Discovery.

Wenqian Li, John R Koethe, Curtis L Gabriel, Heidi J Silver, Timothy J Garrett.

A comprehensive analytical strategy for multiomics analysis of dried blood spots (DBS) has been developed, featuring prespotted stable isotope internal standards (SIIS) for enhanced quantitation. The method combines an optimized Folch extraction protocol for dried plasma spots with high-resolution mass spectrometry analysis using a Thermo Q Exactive Orbitrap mass spectrometer coupled to a Dionex Ultimate 3000 ultrahigh performance liquid chromatography system. This internal quantitative DBS approach addresses key analytical challenges in microsampling applications through strategic incorporation of SIIS directly into the sampling matrix, enabling reliable normalization with improved extraction efficiency. The analytical workflow successfully integrates the simultaneous analysis of metabolites and lipids, demonstrating broad molecular coverage from minimal sample volumes in the microliter range. Method applicability was demonstrated using fasting plasma samples from a cohort of persons with HIV, revealing distinct molecular profiles between diabetic and nondiabetic participants, with hundreds of metabolites and thousands of lipids identified. The analytical platform leverages the inherent advantages of DBS, including simplified sample collection, enhanced stability during storage, and reduced biohazard risk during transportation. This methodology represents a significant advancement in quantitative DBS analysis, particularly valuable for large-scale clinical studies where conventional biobanking presents logistical challenges. The integration of SIIS into the microsampling device establishes a robust foundation for reliable multiomics analysis in resource-limited settings where whole blood or plasma storage is infeasible.

DOI: https://doi.org/10.1021/acs.analchem.5c01713
J Mass Spectrom. 2025 Oct;60(10): e5174

Quantitative UHPLC-MS/MS Analysis of Gemcitabine and Its Active and Inactive Metabolites in Pig Plasma and Lung Tissue in Preparation of Locoregional Therapies for Non-Small Cell Lung Cancer.

Erik Claes, Jeroen M H Hendriks, Thérèse S Lapperre, Johan Van Daele, Andreia Alves, Pieter Annaert, Stijn E Verleden.

  Gemcitabine (dFdC), an effective chemotherapeutic for non-small cell lung cancer (NSCLC), acts through its active metabolite, dFdCTP. Its current pharmacokinetics (PK) knowledge is based on intravenous infusion, which does not represent locoregional administration. In contrast, locoregional studies focus on dFdC, neglecting the role of dFdU (inactive) and dFdCTP, limiting our lung-specific PK insights. Existing dFdCTP assays require lengthy runs and complex protocols, reducing their translatability for large-scale studies. This study presents an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify dFdC, dFdU, and dFdCTP in pig lung tissue and plasma, improving gemcitabine's PK assessment following both systemic and locoregional therapies. A high-throughput UHPLC-MS/MS method was developed and validated for the quantification of dFdC, dFdU, and dFdCTP in pig plasma and lung tissue. Protein precipitation was used, followed by chromatographic separation on a Luna Omega Polar C18 column with a 4-min run time. Linearity was established over the ranges: 0.5-400.0 μg/mL (plasma) and 0.5-400.0 μg/g (lung tissue). It demonstrated robust accuracy, precision, and selectivity with minimal carryover and negligible matrix effects. Stability was confirmed for 4 h at 4°C, 40°C, and on the autosampler. The method's applicability was verified in study samples treated with a 30-min intravenous gemcitabine (1.25 g/m2) infusion. This UHPLC-MS/MS assay overcomes previous methodological limitations by reducing run time and simplifying sample preparation. Its robustness to quantify dFdC, dFdU, and dFdCTP in pig plasma and lung tissue has been confirmed, providing a valuable tool for future PK studies with locoregional therapies for NSCLC.

Keywords:  UHPLC–MS; dFdC; dFdCTP; dFdU; gemcitabine; lung; plasma

DOI:  https://doi.org/10.1002/jms.5174
J Pharm Biomed Anal. 2025 Sep 15. pii: S0731-7085(25)00487-X. [Epub ahead of print]267 117146

Features of sample preparation of cell culture samples for metabolomic screening by LC-MS/MS.

Nikita V Basov, Ekaterina A Butikova, Maria A Sotnikova, Ivan A Razumov, Yulia S Sotnikova, Yuriy V Patrushev, Artem D Rogachev, Nariman F Salakhutdinov, Andrey G Pokrovsky.

  Metabolomic analysis has become an essential tool in the life sciences, providing insights into cellular metabolism. However, preparing cell cultures for metabolomic screening remains challenging, especially with samples containing variable cell numbers. Standardized and reproducible protocols are required to ensure reliable data while maintaining compatibility with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using melanoma cell lines SK-MEL-28 (human) and B16 (mouse) as models, we developed and optimized a convenient sample preparation protocol for metabolomic screening by HPLC-MS/MS. The study is focused on optimizing key steps, including cell lysis, metabolite extraction, and normalization strategies for accurate semiquantitative analysis. The effects of cell count on metabolomic coverage and detection sensitivity were evaluated using hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography. The protocol enables efficient detection of several metabolite classes from samples containing as few as 10,000 cells. The optimal cell count for reliable analysis was found to be 400,000 - 500,000 cells, ensuring consistent and reproducible detection within the method's analytical coverage. Our findings emphasize the importance of cell size and number in metabolomic studies, as larger cells provide improved metabolomic coverage. Moreover, metabolites exhibited varying detection limits, highlighting the need to adjust sample preparation strategies according to metabolite characteristics. The proposed protocol offers a robust and reproducible approach for the metabolomic screening of adherent melanoma cell cultures by HPLC-MS/MS and can be adapted for non-adherent and other cell types. Balancing sensitivity, reproducibility, and feasibility, this method provides a standardized solution for cell metabolomic studies in pharmacometabolomics, cancer research, and related fields.

Keywords:  Cell culture metabolomics; Cell number optimization; HILIC; HPLC-MS/MS standardization; RP LC; Targeted metabolomics

DOI:  https://doi.org/10.1016/j.jpba.2025.117146
bioRxiv. 2025 Sep 09. pii: 2025.09.03.673811. [Epub ahead of print]

MSI.EAGLE: An Open-Source GUI for Streamlined Mass Spectrometry Imaging Analysis.

Daniel J Boehmler, Ashley Woolfork, Ai Wen Tan, Arjun Sengupta, Pinky Kain, Om B Patel, Farheen Akhtar, George McClung, Daniel Ackerman, Terence P Gade, Garret A Fitzgerald, Aalim M Weljie.

Mass Spectrometry Imaging (MSI) is an emergent tool for analyzing spatial molecular distributions, yet data complexity often hinders effective analysis. MSI.EAGLE, an open-source R-Shiny application, makes analysis accessible for non-specialists by integrating advanced tools in a user-friendly interface. The workflow leverages tools from the Cardinal MSI package, with enhanced phenotyping, segmentation, statistical analysis and visualization. The application addresses a gap in spatial biology research by empowering a broader scientific community.

DOI: https://doi.org/10.1101/2025.09.03.673811
Anal Chem. 2025 Sep 16.

Two-Dimensional LC × DMS Analysis of 34 PFAS Compounds.

Christopher R M Ryan, Emir Nazdrajić, J Larry Campbell, Katherine Y Bell, W Scott Hopkins.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous environmental pollutants that pose potential risks to ecosystems and human health. Prior to mass spectrometric analysis of environmental samples, it is necessary to separate PFAS from compounds that can cause ion suppression and compromise analyte identification and quantification accuracy. Although liquid chromatography-mass spectrometry (LC-MS) is the gold standard for PFAS trace analysis, some PFAS species still coelute in the LC dimension and could benefit from an orthogonal dimension of separation. Moreover, an additional orthogonal dimension of separation could potentially aid in the identification of unknown fluorinated species (e.g., those identified within a specified mass-defect range). Here, we investigate the sequential use of LC and differential mobility spectrometry (DMS) separation to analyze 34 PFAS species. Upon incorporating DMS in a two-dimensional (2D) separation scheme, we observed baseline resolution of 29 compounds in the 2D LC × DMS space, with partial resolution of the remaining five. In comparison, only five PFAS compounds were baseline-resolved in 1D LC experiments. Because DMS measurements can be acquired in milliseconds, targeted 2D LC × DMS-MS2 analyses operate on the same time scale as LC-MS2 analysis. However, the limits of quantitation for PFAS using the 2D LC × DMS-MS2 method are slightly higher than those achieved by the state-of-the-art LC-MS2 method owing to ion fragmentation within the energetic DMS environment. Nevertheless, distinct trends observed in the 2D separation space for the various PFAS subclasses will facilitate analyte identification of unknown species in future nontargeted analyses. Finally, we assessed the feasibility of our method for quantifying PFAS in a series of wastewater samples obtained from a Southern Ontario wastewater treatment plant. We were successful in quantifying PFOS, although the concentrations determined were consistently higher than those measured with LC-MS2.

DOI: https://doi.org/10.1021/acs.analchem.5c04119
Anal Chem. 2025 Sep 18.

MassVision: An Open-Source End-to-End Platform for AI-Driven Mass Spectrometry Imaging Analysis.

Amoon Jamzad, Jade Warren, Ayesha Syeda, Martin Kaufmann, Natasha Iaboni, Christopher J B Nicol, John F Rudan, Kevin Y M Ren, David Hurlbut, Sonal Varma, Gabor Fichtinger, Parvin Mousavi.

Mass spectrometry imaging (MSI) combines spatial and spectral data to reveal detailed molecular compositions within biological samples. Despite their immense potential, MSI workflows are hindered by the complexity and high dimensionality of the data, making their analysis computationally intensive and often requiring expertise in coding. Existing tools frequently lack the integration needed for seamless, scalable, and end-to-end workflows, forcing researchers to rely on local solutions or multiple platforms, which hinders efficiency and accessibility. We introduce MassVision, a comprehensive software platform for MSI analysis. Built on the 3D Slicer ecosystem, MassVision integrates MSI-specific functionalities while addressing general user requirements for accessibility and usability. Its intuitive interface lowers barriers for researchers with varying levels of computational expertise, while its scalability supports high-throughput studies and multislide data sets. Key functionalities include visualization, segmentation, colocalization, data set curation, data set merging, spectral and spatial preprocessing, statistical analysis, AI model training, and AI deployment on full MSI data. We detail the workflow and functionalities of MassVision and demonstrate its effectiveness through different experimental use cases such as exploratory data analysis, ion identification, and tissue-type classification on in-house and publicly available data from different MSI modalities. These use cases underscore MassVision's ability to seamlessly integrate MSI data handling steps into a single platform and highlight its potential to reveal new insights and structures when examining biological samples. By combining cutting-edge functionality with user-centric design, MassVision addresses longstanding challenges in MSI data analysis and provides a robust tool for advancing the user's ability to achieve biologically meaningful insights from MSI data. MassVision is freely available via 3D Slicer (documentation: https://SlicerMassVision.readthedocs.io/). The in-house MSI data have been made publicly available in MetaboLights with the identifier MTBLS12868.

DOI: https://doi.org/10.1021/acs.analchem.5c04018
Nat Methods. 2025 Sep 15.

MSnLib: efficient generation of open multi-stage fragmentation mass spectral libraries.

Corinna Brungs, Robin Schmid, Steffen Heuckeroth, Aninda Mazumdar, Matúš Drexler, Pavel Šácha, Pieter C Dorrestein, Daniel Petras, Louis-Felix Nothias, Václav Veverka, Radim Nencka, Zdeněk Kameník, Tomáš Pluskal.

Untargeted high-resolution mass spectrometry is a key tool in clinical metabolomics, natural product discovery and exposomics, with compound identification remaining the major bottleneck. Currently, the standard workflow applies spectral library matching against tandem mass spectrometry (MS2) fragmentation data. Multi-stage fragmentation (MSn) yields more profound insights into substructures, enabling validation of fragmentation pathways; however, the community lacks open MSn reference data of diverse natural products and other chemicals. Here we describe MSnLib, a machine learning-ready open resource of >2 million spectra in MSn trees of 30,008 unique small molecules, built with a high-throughput data acquisition and processing pipeline in the open-source software mzmine.

DOI: https://doi.org/10.1038/s41592-025-02813-0
Rapid Commun Mass Spectrom. 2025 Dec 30. 39(24): e10141

Comprehensive Metabolic Profiling of Resigratinib, a Novel FGFR Inhibitor, Using Integrated LC-MS/MS and LC-Orbitrap-HRMS.

Xiaoxia An, Yanting Mao, Ali Fan, Ting Ma.

   RATIONALE: Resigratinib, a potent fibroblast growth factor receptor (FGFR) inhibitor, is under clinical development for solid tumors such as cholangiocarcinoma. However, data on its hepatic metabolism remain limited. To support further development, this study aimed to characterize its in vitro metabolism using rat, dog, monkey, and human liver microsomes.
METHODS: A sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated for quantifying resigratinib in liver microsomes. Metabolite characterization was performed using LC coupled with benchtop Orbitrap high-resolution mass spectrometry (LC-Orbitrap-HRMS) in full-scan MS/dd-MS2 and parallel reaction monitoring (PRM). This approach enabled accurate mass measurement, chemical formula assignment, and structural elucidation via MS2 fragmentation interpretation.
RESULTS: The established method exhibited excellent linearity over the concentration range of 1.0-1000 nM. Resigratinib displayed low clearance in dog (t1/2 = 91.2 min), intermediate clearance in rat (t1/2 = 20.2 min), and high clearance in monkey (t1/2 = 6.8 min) and human (t1/2 = 14.0 min) systems. Ten metabolites were identified, with M3 (bis-demethylation), M5 (O-demethylation), and M9 (N-demethylation) identified as the major metabolites. Recombinant human cytochrome P450 enzyme analysis and chemical inhibition studies indicated that CYP3A4 is the predominant enzyme responsible for resigratinib metabolism.
CONCLUSION: This study presents the first integrated analytical approach, combining LC-MS/MS and LC-Orbitrap-HRMS, for the in vitro metabolic assessment of resigratinib. The observed metabolic profiles provide an essential foundation for further toxicological and clinical investigations.

Keywords:  CYP3A4; fibroblast growth factor receptor; high‐resolution mass spectrometry; metabolic profiles; resigratinib

DOI:  https://doi.org/10.1002/rcm.10141
Anal Chem. 2025 Sep 15.

Efficient and Wide Chemical-Space Ionization of Organic Contaminants Using LC-MS with a Miniaturized Plasma Source Applying Different Discharge Gases.

Irene Caño-Carrillo, Bienvenida Gilbert-López, David Moreno-González, Joachim Franzke, Juan F García-Reyes.

Dielectric barrier discharge ionization has gained significant interest due to its versatility and broad chemical coverage. Although electrospray ionization (ESI) is the most commonly used ionization source for organic contaminant analysis by liquid chromatography-mass spectrometry (LC-MS), it has limitations such as low ionization efficiency for nonpolar compounds and matrix effects. This study investigates the potential of flexible microtube plasma (FμTP) as an alternative ionization source for the LC-MS determination of multiclass pesticides comprising ESI-amenable and organochlorine contaminants. The analytical performance of FμTP was assessed in terms of limits of quantification, reproducibility, linearity, and matrix effects, comparing the results to those obtained with ESI and atmospheric pressure chemical ionization (APCI) sources. Sensitivity assessment based on calibration slopes showed that 70% of the pesticides had higher sensitivity with FμTP than with ESI. Regarding the matrix effects, between 76 and 86% of the pesticides showed negligible matrix effects for FμTP, compared to 35-67% for ESI and 55-75% for APCI across the different matrices evaluated. The study further explored the use of argon and argon-propane mixtures as alternatives to helium as discharge gases. Results showed similar LOQs for nearly 90% of the pesticides in the positive mode and 80% of the organochlorines in the negative mode. Notably, some ion species differed when using argon-based gases for certain organochlorine pesticides, suggesting the discharge gas influences the ionization mechanism, especially in the negative mode. Overall, FμTP proves to be a sensitive and robust miniaturized ionization source, expanding the chemical space and making it useful for both target and nontarget screening applications.

DOI: https://doi.org/10.1021/acs.analchem.5c03745
Nucleic Acids Res. 2025 Sep 05. pii: gkaf895. [Epub ahead of print]53(17):

Toward standardized epitranscriptome analytics: an inter-laboratory comparison of mass spectrometric detection and quantification of modified ribonucleosides in human RNA.

Martin Hengesbach, Chi-Kong Chan, Tulsi Bhandari, Alan Bruzel, Michael S DeMott, Ganna Podoprygorina, Guangxin Sun, Ellen Tabeling, Vivian G Cheung, Peter C Dedon, Mark Helm, Patrick A Limbach.

The human RNome comprises all forms of RNA and the 50 + chemical structures-the epitranscriptome-that modify them. Understanding the diverse functions of RNA modifications in regulating gene expression and cell phenotype requires technologies such as RNA sequencing-based modification mapping and mass spectrometry-based quantification of modified ribonucleosides. Liquid chromatography-coupled tandem quadrupole mass spectrometry (LC-MS/MS) is the gold standard for detecting and quantifying modified ribonucleosides with accuracy and precision. However, variations in RNA isolation, processing, and LC-MS/MS analysis have hindered reproducibility across laboratories, which is essential for accurate quantification of RNA modifications. As guidance toward harmonization, we report a multi-laboratory comparison of workflows for LC-MS/MS RNA modification analysis. We compared protocols for sample shipment, RNA hydrolysis, LC-MS/MS analysis, and data processing among three laboratories working with the same total RNA samples. We detected and quantified 17 modifications consistently across protocols and operators, with another 7 that were sensitive to experimental conditions, reagent contamination, and ribonucleoside instability, leading to poor precision among laboratories. Agreement among the three labs was strong, with coefficients of variation of 20% and 10% for relative and absolute quantification, respectively. These findings establish a robust and readily adoptable epitranscriptome analytical platform that enables reliable comparisons across laboratories.

DOI: https://doi.org/10.1093/nar/gkaf895
Clin Biochem. 2025 Sep 13. pii: S0009-9120(25)00142-0. [Epub ahead of print] 111013

Reliable LC-MS/MS method development and validation for the determination of methylmalonic acid, methylcitric acid, malonic acid, ethylmalonic acid, and total homocysteine in dried blood spots.

Chunyan Zhang, Nan Bai, Yanling Yang, Yuxuan Cheng, Xiyu He, Meng Wang, Honghui Zhou, Yaping Tian.

   OBJECTIVES: Metabolic disorders in newborns can cause significant morbidity and mortality if not diagnosed and managed early. In this study, we developed and validated a reliable LC-MS/MS method to simultaneously quantify methylmalonic acid, methylcitric acid, malonic acid, ethylmalonic acid, and total homocysteine associated with metabolic disorders.
DESIGN AND METHODS: According to Clinical Laboratory Standards Institute (CLSI) guidelines, various analytical performances (i.e., precision, linearity, accuracy and reference intervals) were rigorously evaluated. Meanwhile, sample preparation, chromatographic separation and mass spectrometric detection and incorporating stable isotope-labeled internal standards were optimized. Furthermore, calibrators and quality controls were developed to ensure standardization and traceability.
RESULTS: The coefficient of variations for precision were less than 10.0 %. Meanwhile, the results showed high accuracy (recoveries: 94.57 %-109.60 %) and robust linearity (R2 > 0.9935) over clinically relevant ranges. The limits of detection and limits of quantification for all analytes were established, enabling sensitive detection of pathological elevations. Reference intervals for pediatric populations (ages 0-1 month and 2 months to 18 years) were determined, providing essential baselines for clinical interpretation.
CONCLUSIONS: The newly developed method demonstrated good analytical performance and offers a standardized, high-throughput solution for clinical laboratories to improve early diagnosis and personalized management of metabolic diseases.

Keywords:  Dried blood spots; Ethylmalonic acid; Homocysteine; LC-MS/MS; Malonic acid; Methylcitric acid; Methylmalonic acid

DOI:  https://doi.org/10.1016/j.clinbiochem.2025.111013
ACS Omega. 2025 Sep 09. 10(35): 39463-39470

First Specific Analytical Method for Quantification of Halide Ions by UHPLC-MS Using Reverse Phase Chromatography C18.

Steven Bonert, Hervé Sayer, Ghislaine Tabone, Benoit Robert.

In the pharmaceutical industry, optimizing methods for trace detection and stoichiometry determination of salts is highly sought after, particularly for studies on pharmaceutical compounds in the development phase. Current methods for halide ion detection are commonly carried out using ion chromatography (IC) due to the high specificity and sensitivity of this technique, which allows the detection of various ions at very low concentrations. Advanced techniques such as mass spectrometry, which offers significantly higher sensitivity thanks to its specificity, can be particularly advantageous in analytical applications both for trace detection with a view to quantification and for its accuracy in stoichiometric calculations, allowing the precise determination of the ratio between the compound of interest and the counterion. To our knowledge, no LC-MS methods using a reverse-phase polarity chromatographic system (C18), commonly employed in high-performance liquid chromatography (HPLC) or ultrahigh-performance liquid chromatography (UHPLC), currently exist that allow for sufficiently effective halogen separation for accurate quantification. We present a new rapid and efficient method compliant with International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use standards using a C18 reverse phase, which enables optimal separation of three halogens in order to precisely quantify analytes through mass spectrometry. This method will be illustrated with a relevant application, dedicated to the stoichiometry investigation of pharmaceutical compounds in the form of salts or halide ion residues.

DOI: https://doi.org/10.1021/acsomega.4c10817
RSC Adv. 2025 Sep 11. 15(40): 33515-33521

Single-cell metabolite annotation by tandem mass spectrometry imaging and ab initio molecular dynamics-based fragmentation.

Mateo Topalović, Ivana Marković, Vlatka Periša, Maja Lukić, Ema Pavičić, Iva Lukić, Stefan Mrđenović, Igor Lukačević, Željko Debeljak.

Reliable single-cell mass spectrometry (MS) imaging and metabolite annotation represent a challenge due to small cellular dimensions, small amount of desorbed materials and densely populated databases of the m/z values of endogenous compounds. To resolve these issues, a highly sensitive analytical approach was devised for metabolite annotation purposes, relying on the correspondence between the m/z values from single-cell MS/MS spectra and the m/z values of the molecular fragments calculated using ab initio molecular dynamics (AIMD). The approach was applied to the annotation of m/z 337.11 Da, which cellular content is increased in chronic lymphocytic leukemia (CLL). To evaluate the approach, five candidate compounds were selected by a metabolite database search using the given m/z. Matrix-assisted laser desorption/ionization ion trap-time-of-flight (MALDI IT-TOF) MS/MS spot analysis of S-nitrosoglutathione (GSNO), one of the candidate compounds, preceded single-cell MS/MS imaging: five fragments were present in the empirical and in silico spectra of the GSNO solution. The sensitivity of single-cell MS imaging was optimized, and MS/MS spectra were recorded for different lymphocytes containing 0-3 fragments that were present in the in silico spectra of three glutathione-related compounds; however, there was no match between the empirical and in silico spectra of the other candidate compounds. The lateral distribution of the selected fragment showed ∼3 μm shift with respect to the optical image of the lymphocyte. The novel concept developed for single-cell MS imaging enabled metabolite annotation in malignant lymphocytic clones and showed potential for metabolite annotation in other cell suspensions.

DOI: https://doi.org/10.1039/d5ra05470b
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Sep 10. pii: S1570-0232(25)00340-X. [Epub ahead of print]1267 124786

Development and validation of general urine screening method for performance enhancing drugs in racehorses utilizing liquid chromatography-high resolution mass spectrometry (LC-HRMS).

Saurabh Dubey, Izabela Lomnicka, Pamela Waller, Dharmikkumar Vora, Levent Dirikolu.

  The complexity of the drug market and the constant updating of drugs have been challenging issues for drug regulatory authorities. In this manuscript, a high-throughput automated assay based on Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) suitable for use as an initial testing procedure covering multiple classes of compounds prohibited in horse racing is described. The assay requires a 250-μL urine aliquot, which is subjected to enzymatic hydrolysis followed by Biotage Isolute supported liquid extraction plates using Biotage Extrahera system, evaporation, and reconstitution in a 96-well collection plate. LC-HRMS analyses were carried out on a Thermo Fisher Q-Exactive Mass spectrometer (equipped with HESI source interface) coupled with Vanquish UHPLC system linked to ACE Excel column. Drug targets were identified by retention time and accurate mass, with a mass tolerance window of +/- 5 ppm. The screening method was validated for over 250 drug targets and/or their metabolites in a 7-min run. Validation data including sensitivity, specificity, extraction recovery and precision are presented. As the method employs full-scan mass spectrometry, an unlimited number of drug targets can theoretically be incorporated.

Keywords:  Doping; Equine; High resolution; Mass spectrometry; Urine

DOI:  https://doi.org/10.1016/j.jchromb.2025.124786
J Chromatogr A. 2025 Sep 11. pii: S0021-9673(25)00717-4. [Epub ahead of print]1762 466374

A complementary multi-aptamer approach for efficient extraction of steroid hormones and application to their determination in biological samples using Q-TOF LC-MS.

Xijia Zhang, Yingjie Zhuo, Qianqian Xu, Ningning Wang, Hao Zhong, Dongsheng Li, Juanjuan Zhao.

  Steroid hormones play a crucial role in human physiological functions, and the simultaneous quantification of multiple steroid hormones in biological matrices has considerable clinical relevance. However, the simultaneous quantification of steroid hormones in biological matrices remains analytically challenging due to their ultra-trace concentrations and substantial matrix interference. A novel complementary multi-aptamer strategy was proposed in our study. Multi-aptamer magnetic beads (MA-MBs) were synthesized by simultaneously immobilizing estrogen-specific aptamer (AptE) and corticoid-specific aptamer (AptC) on the magnetic beads. Then MA-MBs was used to extract multiple steroid hormones in biological matrices based on the combined specificity of complementary aptamers. Following extraction, the enriched steroid hormones are quantified using quadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC-MS). MA-MBs demonstrated a rapid magnetic response (5 s), high extraction efficiency, and excellent reusability (at least 10 cycles). Under optimal extraction conditions, the proposed MA-MBs-Q-TOF LC-MS method exhibited a wide linear range (0.02-200 ng/mL), low limits of detection (0.02-1 ng/mL), and high recovery rates (92.93 %-104.93 %) for 10 types of steroid hormones. Finally, the validated method was applied to quantify ten clinically-relevant steroid hormones in serum samples, demonstrating its potential for advanced endocrine research applications. Furthermore, this study provides a robust platform for multiplexed targets analysis in complex biological matrices.

Keywords:  Affinity magnetic bead technology; Biological samples; Liquid chromatography-mass spectrometry; Multi-aptamer technology; Steroid hormones

DOI:  https://doi.org/10.1016/j.chroma.2025.466374
J Pharm Biomed Anal. 2025 Sep 08. pii: S0731-7085(25)00486-8. [Epub ahead of print]267 117145

Validation and quantitation of six neurotransmitters in rat plasma and brain using liquid chromatography quadrupole-time-of-flight mass spectrometry.

Laerissa Reveil, Maria Lasaosa, Amanda Furman, A M Iqbal O'Meara, Matthew S Halquist.

  Delirium is a cognitive dysfunction observed following sedation of critically injured patients. Neurotransmitters are endogenous chemical messengers that play key roles in a variety of essential nervous system functions, and their dysregulation is believed to contribute to the development of delirium. Monitoring neurotransmitters such as acetylcholine, dopamine, serotonin, gamma-aminobutyric acid, gamma-hydroxybutyric acid, and glutamic acid for biomarkers of delirium could improve our understanding of delirium pathogenesis and provide evidence for more efficacious treatments. However, current methods have limited scopes of analysis, require large sample volumes, or use complicated extraction methods. In this study, a novel, analytical approach was developed using liquid chromatography quadrupole-time-of-flight mass spectrometry to measure neurotransmitters in limited sample. This method was used to measure neurotransmitters in plasma and brain tissue samples taken from rats treated with a benzodiazepine sedative and opioid analgesic, a drug combination commonly prescribed in intensive care units and known to precipitate delirium. The analytical method was validated for selectivity, matrix effect, accuracy and precision, dilution integrity, and stability in rat plasma and partially validated in rat brain. The calibration curve ranged from 10 to 5000 ng/mL across the analytes. Within-run and between-run bias and precision of the LLOQ and QCs in plasma and brain were within 20 % and 15 %, respectively. Analytes were stable at 2 ºC and -80 ºC. Extraction recoveries from a simple protein precipitation ranged from 85 % to 133 %. The method was successfully applied to measure analytes in rat brain homogenates from a delirium model study.

Keywords:  Bioanalysis; HILIC column chemistry; High resolution mass spectrometry; Neurotransmitters

DOI:  https://doi.org/10.1016/j.jpba.2025.117145
Environ Sci Technol Lett. 2025 Sep 09. 12(9): 1162-1168

A Novel Chemical-Space-Dependent Strategy for Compound Selection in Non-target LC-HRMS Method Development Using Physicochemical and Structural Data.

Lapo Renai, Viktoriia Turkina, Tobias Hulleman, Alexandros Nikolopoulos, Andrea F G Gargano, Elvio D Amato, Massimo Del Bubba, Saer Samanipour.

  The virtual chemical space of substances, including emerging contaminants relevant to the environment and exposome, is rapidly expanding. Non-targeted analysis (NTA) by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is useful in measuring broad chemical space regions. Internal standards are typically used to optimize the selectivity and sensitivity of NTA LC-HRMS methods, assuming a linear relationship between structure and behavior across all analytes. However, this assumption fails for large, heterogeneous chemical spaces, narrowing measurable coverage to structurally similar compounds. We present a data-driven strategy for unbiased sampling of candidate structures for NTA LC-HRMS method development from extensive chemical spaces, such as the U.S. EPA's CompTox (>1 million chemicals). The workflow maximizes physicochemical/structural diversity using precomputed PubChem descriptors (e.g., molecular weight, XLogP) and grants LC-HRMS compatibility thanks to predicted mobility and ionization efficiency from molecular fingerprints. The resulting measurable compound lists (MCLs) provide broad, heterogeneous coverage for NTA method development, validation, and boundary assessment. Applied to the CompTox space, the approach yielded MCLs with greater chemical coverage and broader predicted LC-HRMS applicability than conventional "watch list" contaminants, offering a robust framework for enhancing NTA's measurable chemical space while preserving diversity.

Keywords:  Chemical Space; Emerging Contaminants; Exposomics; Ionization Efficiency; Liquid Chromatography; Mass Spectrometry; Mobility; Non-target Analysis

DOI:  https://doi.org/10.1021/acs.estlett.5c00759
RSC Adv. 2025 Sep 11. 15(40): 33536-33548

Inner-wall biochar-coated pipette tip microextraction for rapid and sustainable determination of tyrosine kinase inhibitors in plasma.

Xiaofei Zhang, Mengyuan Lv, Yuyang Wang, Nan Zhang, Di Chen.

Tyrosine kinase inhibitors (TKIs) are critical in cancer therapy, but their clinical application is complicated by narrow therapeutic windows and significant interpatient variability in pharmacokinetics. To address this, this study presents an effective pipette-tip solid-phase microextraction (PT-μSPE) method using kapok fiber-derived biochar (KFB) as a green and cost-effective sorbent for the extraction of TKIs in plasma. The KFB was incorporated as an inner-wall coating within a standard pipette tip, minimizing backpressure and enhancing automation compatibility. This PT-μSPE method is optimized for efficiency and sensitivity, with ethanol used as the desorption solvent. The method is coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of TKIs, demonstrating excellent linearity (R 2 > 0.99), sensitivity (LOD: 0.022-0.125 ng mL-1), and reproducibility. Furthermore, the method exhibits notable green chemistry characteristics, including minimal solvent usage and environmental sustainability. The proposed method offers a practical, scalable solution for TKI quantification for potential clinical and pharmacokinetic studies, with potential for high-throughput integration into automated systems.

DOI: https://doi.org/10.1039/d5ra05405b
Anal Methods. 2025 Sep 17.

A UPLC-MS/MS method for the determination of 2-nitro-1,3-propanediol, a metabolite of Bronopol, in human urine.

Isabell Schönrath, Christoph Schmidtkunz, Katja Küpper, Edgar Leibold, Gabriele Leng.

Bronopol is a preservative with applications in various industrial fields; thus, the general population may be exposed to this substance. For the development of an analytical method for human biomonitoring (HBM) in urine, 2-nitro-1,3-propanediol was postulated as a suitable biomarker according to findings in animal studies. The herein described UPLC-MS/MS method enables the quantification of 2-nitro-1,3-propanediol in a concentration range from 0.5 μg l-1 (LOQ) to 5000 μg l-1. The sample preparation is based on a liquid-liquid extraction with ethyl acetate. Analyte losses are compensated by the authentic, isotope-labelled internal standard 2-nitro-1,3-propanediol-13C3. The method fulfils all reliability criteria: intra- and interday coefficients of variation (CVs) were determined in spiked pooled urine samples at 1, 10 and 100 μg l-1 and did not exceed 5%. The corresponding accuracies were between 104 and 105%. In individual urine samples (robustness, n = 10) spiked at the same levels, CVs were <8% and accuracies ranged between 98 and 104%. Applying different hydrolysis protocols, we could rule out any phase II metabolites of 2-nitro-1,3-propanediol from being excreted in urine. Thanks to samples from a human metabolism study involving oral intake of Bronopol, we were able to confirm the postulated metabolite. In representative specimens, the analyte could be quantified in large amounts, demonstrating the applicability of the method both for high concentrations in metabolism studies and occupational medicine, and at lower levels, for example, in field studies to assess the burden of the general population.

DOI: https://doi.org/10.1039/d5ay00960j
Anal Methods. 2025 Sep 17.

A thorough LC-MS/MS strategy to quantify abaloparatide in rat plasma: method development, validation and application.

Ying Kong, Xiangping Li, Yan Li, Yuan Tan, Tongfang Li, Yani Xiao, Li Zhang, Qinglong Yu, Rong Rong.

BACKGROUND: Abaloparatide is a newly approved analog of parathyroid hormone-related peptide. It is used for the treatment of osteoporosis in postmenopausal women who have a high risk of fracture. The aim of our work was to establish a novel liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method to achieve rapid, sensitive and robust quantification of abaloparatide in biological samples.
RESULTS: Plasma samples were purified by protein precipitation, and abaloparatide was then chromatographically separated on a positively charged surface hybrid column, which offered reliable chromatographic results without the addition of any strong ion-pairing reagents. Ion transitions of m/z 566.512 (7+) → 632.488 for abaloparatide and 687.05 (6+) → 787.26 for the internal standard (teriparatide) were selected for multiple-reaction mass spectrometry detections. The method was validated over a range of 0.02 to 8 ng mL-1 and was further demonstrated to be accurate and precise. Meanwhile, the method presented acceptable recoveries for abaloparatide (88.7-103%) and the internal standard (102%), and there was no significant matrix effect. Abaloparatide was proved to be stable under the conditions of 4 °C for 2 h at -80 °C for 50 days and three freeze-thaw cycles (-80 °C to 4 °C). The method was successfully applied to the pharmacokinetic study of abaloparatide in rats after subcutaneous administration.
SIGNIFICANCE: To the best of our knowledge, this is the first mass spectrometry-based method developed to quantify abaloparatide in plasma samples. It not only offers an attractive choice for abaloparatide development but may also promote the application of LC-MS/MS in peptide bioanalysis.

DOI: https://doi.org/10.1039/d5ay01189b
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Sep 14. pii: S1570-0232(25)00345-9. [Epub ahead of print]1267 124791

Eco-friendly microextraction technique using SFO-DLLME coupled with HPTLC for plasma analysis of metformin and imeglimin.

Venkata Suresh Ponnuru, D Renuka, M Siva Prasad, R Hari Prasad Reddy, M Bhargava Reddy, K N Rajani Kanth, G Naga Raju, N Rama Rao, M Hemanth Kumar.

  This study aimed to develop and validate a green, sensitive, and high-throughput analytical method for the simultaneous determination of metformin and imeglimin in plasma samples using gliclazide as an internal standard. A novel microextraction technique was proposed, combining salt-induced dispersive liquid-liquid microextraction based on solidified floating organic droplet (SI-DLLME-SFOD) with high-performance thin-layer chromatography HPTLC-densitometric detection. This approach significantly improves greenness, sensitivity, and simplicity compared to conventional extraction and chromatographic techniques. The method was optimized for maximum resolution using silica gel 60 F254 pre-coated TLC plates as the stationary phase and a mobile phase comprising methanol: chloroform: 0.5 % w/v ammonium acetate (8:1:1, v/v/v). Detection was performed at 254 nm. The method was validated following US FDA bioanalytical guidelines. The Rf values were observed at 0.45 ± 0.04 for imeglimin, 0.63 ± 0.04 for metformin, and 0.84 ± 0.04 for gliclazide. The method exhibited excellent linearity in the range of 100-1000 ng/band, with correlation coefficients (r2) exceeding 0.998. The average recoveries were 90.91 % for imeglimin and 92.61 % for metformin, with RSD values of 0.42 and 0.39, respectively, confirming high precision and accuracy. The SI-DLLME-SFOD protocol enables efficient extraction, lower solvent consumption, and improved analyte enrichment. The developed method was evaluated using green analytical chemistry metrics and its eco-friendliness was confirmed. It demonstrated reduced solvent usage, minimal waste generation, and a lower environmental burden, establishing its superiority over previously reported sample preparation techniques. Furthermore, the validated method was successfully applied to real samples (human plasma) to assess the concentration, thereby demonstrating the potential value of the method for routine use. This method provides a green, efficient, and validated strategy for the bioanalysis of antidiabetic drugs in human plasma, supporting its potential application in routine clinical diagnostics and therapeutic drug monitoring.

Keywords:  Green chemistry metrics; HPTLC; Imeglimin; Metformin; Microextraction; Salting-out assisted liquid-liquid extraction

DOI:  https://doi.org/10.1016/j.jchromb.2025.124791
Nanoscale Adv. 2025 Sep 11.

Advances in the chemical analysis of nitrite in environmental and biological samples.

Nadeen Rajab, Hosny Ibrahim, Daohong Zhang, Ahmed F A Youssef, Rabeay Y A Hassan.

Nitrite, a potential environmental pollutant, poses a significant threat to human health. Thus, accurate and sensitive detection methods are essential for effective continuous monitoring and surveillance. In this regard, a wide range of instrumental methods for the precise determination of nitrite in different types of complex samples is collected and discussed. Besides the classical methods, chromatographic and spectroscopic techniques are included. Although these methods exhibit high sensitivity and selectivity, they involve high cost and complicated operating protocols, and warrant high caution in sample preparations. Other reported techniques, such as electrochemical and bio-electrochemical methods, could offer onsite detection and disposability, and involve handheld devices. Such features are required for simple optimization, field applicability for analysis of a large number of samples, fast response, simple device calibration, and validation. Thus, nanostructure-based electrochemical approaches are widely developed and applied in the analysis of target analytes in complex biological and environmental matrices using a few microlitres of the samples, without any prior sample preparation. Therefore, to consider the global market's needs, challenges, and perspectives on each reported method for nitrite, a comprehensive discussion has been included in this review.

DOI: https://doi.org/10.1039/d5na00503e