bims-metlip 2025-11-16 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–11–16
twenty-two papers selected by
Sofia Costa, Matterworks

Integrative Metabolic-Flux Platform for Analysis, Contextualization, and Targeting (IMPACT): Bridging Untargeted and Targeted Flux Analysis.
Determination of Gnetol in Murine Biological Matrices by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): Application in a Biodistribution Study.
A novel derivatization reagent for ultra-sensitive determination of free fatty acids in complex matrices by UHPLC-QQQ-MS.
Enhancing Hydroxyl Metabolite Analysis through In Situ Derivatization-Enabled Desorption Electrospray Ionization-Mass Spectrometry.
A Novel UPLC-MS/MS Method for Determining Tegoprazan in Rat Plasma: An Application in a Rat Pharmacokinetic Study.
Matrix effects in untargeted LC-MS metabolomics: From creation to compensation with post-column infusion of standards.
Determination of residual 15 quaternary ammonium compounds in dairy products by dispersive solid-phase extraction purification with ultra-high performance liquid chromatography-tandem mass spectrometry.
Sensitive and Selective Quantification of Acrylamide in French Fries and Canola Oil by Fluorous Derivatization with Liquid Chromatography-Tandem Mass Spectrometry.
Optimizing MS Parameters for Data-Independent Acquisition (DIA) to Enhance Untargeted Metabolomics.
Simultaneous determination of 19 Aconitum alkaloids in whole blood and urine by ultra-high performance liquid chromatography-tandem mass spectrometry.
Rapid LC-MS/MS-based quantitative serum tryptophan metabolomics platform identifies and monitors novel biomarkers for diabetic kidney disease.
Stability of 22 Sedative-Type Drugs and Metabolites in Human Urine under Variable pH, Temperature, and Freeze-Thaw Conditions.
Development of an LC-MS/MS method for the quantification of sunitinib and its metabolites in human nail and skin.
Quantitation of Plasma Bedaquiline Concentrations in Indian MDR-TB Patients Using Liquid Chromatography-Tandem Mass Spectrometry.
UHPLC-MS/MS for Antipsychotic Drug Monitoring: A Systematic Review of Clinical and Analytical Performance.
Development of an effective method for biomonitoring of per- and polyfluoroalkyl substances and phthalate diesters and monoesters in human hair using liquid chromatography coupled with tandem mass spectrometry.
Automated magnetic bead‑based LC-Triple Quadrupole MS approach for sensitive colistin therapeutic drug monitoring in human plasma.
Development and Validation of a QuEChERS-Based LC-MS/MS Method for Natamycin in Imported Agricultural Commodities in Korea.
A systematic review of organophosphorus pesticides detection in biological matrices.
Sequential Detection of Biomolecules in Formalin-fixed, Paraffin-embedded Samples with Mass Spectrometry Imaging.
Integrated Analytical System for Comprehensive Two-Dimensional Enantio-Gas Chromatography and Low-Pressure Gas Chromatography Mass Spectrometry Utilizing a Switching Valve: Design and Optimization.
Applications of metabolomic techniques to explore the diversity of phenolic compounds in pseudocereals: Effects of processing and health implications.

Bioinformatics. 2025 Nov 14. pii: btaf591. [Epub ahead of print]

Integrative Metabolic-Flux Platform for Analysis, Contextualization, and Targeting (IMPACT): Bridging Untargeted and Targeted Flux Analysis.

Collin Starke, Janneke Hendriks, Judith Wahrheit, Frank Fluitman, Jules Beekwilder, Florence Miramella Schempp, Karsten Hiller.

   SUMMARY: The "Integrative Metabolic-Flux Platform for Analysis, Contextualization, and Targeting" (IMPACT) is a comprehensive, fully modular platform designed for both targeted and untargeted metabolomics analysis in stable-isotope labeling experiments. It facilitates the accurate calculation of mass isotopomer distributions (MIDs) and the annotation of unknown metabolites with a contextualization algorithm, addressing the challenges in metabolomics research. IMPACT integrates an entire preprocessing pipeline for LC-MS data, including peak picking, feature grouping, and peak filling, along with advanced features for isotope detection, MID calculation, and its core feature contextualization, enabling metabolite integration into biological pathway networks. The platform supports various file formats and offers user-friendly online access, making it accessible for researchers seeking to elucidate metabolic pathways and networks with precision and reliability.
AVAILABILITY AND IMPLEMENTATION: IMPACT is implemented in Python 3.9 and R 4.3.2, with a front-end in Javascript utilizing the Cytoscape.js library for data visualization. It is available as a docker container and can be accessed online at https://impact.bioinfo.nat.tu-bs.de, providing a user-friendly interface for metabolomics data analysis.

Keywords:  LC-MS; Labeling; MIDs; Metabolomics; Targeted; Untargeted

DOI:  https://doi.org/10.1093/bioinformatics/btaf591
Int J Mol Sci. 2025 Oct 24. pii: 10358. [Epub ahead of print]26(21):

Determination of Gnetol in Murine Biological Matrices by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): Application in a Biodistribution Study.

Boyu Liao, Hongrui Jin, Huan Chen, Yuxin Zhang, Xuexian Deng, Jingyi Yao, Na Li, Shaoshu Xu, Jingbo Wang, Mingming Gao, Xiaoying Zhang, Paul C L Ho, Hui Liu, Hai-Shu Lin.

  Gnetol (trans-2,3',5',6-tetrahydroxystilbene), a naturally occurring stilbene structurally related to resveratrol (trans-3,5,4'-trihydroxystilbene; RES), has been reported to possess multiple health-promoting activities. In order to support its potential nutraceutical application, a reliable chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the quantitative determination of gnetol in mouse plasma and tissue samples, using isotopically labeled RES-13C6 serving as the internal standard (IS). Electrospray ionization (ESI) was performed in negative mode, with multiple reaction monitoring (MRM) transitions m/z 243.2 → 175.0 for gnetol and m/z 233.1 → 191.0 for the IS. Chromatographic separation was achieved on a reversed-phase HPLC column using a 5-min gradient delivery of acetonitrile and 2 mM ammonium acetate at 0.5 mL/min and 40 °C. The linear calibration curve covered the concentration range of 5.0-1500 ng/mL, and the method validation confirmed its selectivity, accuracy, precision, stability, and dilution integrity. The developed method was subsequently applied to a biodistribution study in mice after oral administration of gnetol at 400 µmol/kg (equivalent to 97.7 mg/kg). Gnetol was rapidly absorbed and extensively distributed in key pharmacologically relevant organs. Despite its poor aqueous solubility, oral uptake was not significantly hindered. Collectively, these findings demonstrate that gnetol exhibits favorable absorption and tissue distribution profiles, supporting its promise as a candidate for nutraceutical development.

Keywords:  LC–MS/MS; biodistribution; gnetol; nutraceutical; resveratrol

DOI:  https://doi.org/10.3390/ijms262110358
Talanta. 2025 Nov 07. pii: S0039-9140(25)01595-4. [Epub ahead of print]299 129104

A novel derivatization reagent for ultra-sensitive determination of free fatty acids in complex matrices by UHPLC-QQQ-MS.

Yuying Lin, Yiran Zhao, Chengfeng Gao, Haoyuan Zeng, Yue Zhuo, Yi Zhun Zhu, Na Li, Jian-Lin Wu, Xiqing Bian.

  Plant oils are vital dietary sources of ω-3, ω-6, and ω-9 fatty acids, but their accurate quantification remains challenging due to their low abundance and complex matrix interference. Herein, we developed a novel derivatization method using 1-(2-diisopropylaminoethyl) piperazine (DPATP) coupled with ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS). This method offers the simultaneous analysis of 11 fatty acids with a remarkable 300-fold sensitivity enhancement, and limits of quantification as low as 0.4 fg. Comprehensive optimization revealed that liquid-liquid extraction (LLE) outperformed solid-phase extraction (SPE) in recovery efficiency. The derivatization reaction (30 °C for 10 min) was optimized to improve chromatographic resolution and ionization efficiency. Method validation confirmed good linearity, high precision, and acceptable accuracy. Minimal matrix effects and satisfactory recoveries further underscored the method's robustness. The established derivatization-UHPLC-QQQ-MS/MS approach was successfully applied to quantify FFAs in plant oils. This DPAPT-derivatization LC-MS platform is a powerful tool for ultra-sensitive FFA analysis, promising broad applicability in food science, lipidomics, clinical diagnostics, and environmental analysis.

Keywords:  Analytical method development; Derivatization-LC-MS; Fatty acid profiling; Mass spectrometry; Ultra-sensitive detection

DOI:  https://doi.org/10.1016/j.talanta.2025.129104
Anal Chem. 2025 Nov 14.

Enhancing Hydroxyl Metabolite Analysis through In Situ Derivatization-Enabled Desorption Electrospray Ionization-Mass Spectrometry.

Yen-Chu Lin, Guan-Yuan Chen, Ya-Jin Jheng, Hsiao-Wei Liao.

Unlocking the full potential of metabolomics hinges on significantly improving the detection of metabolites, particularly those containing hydroxyl groups, which often remain challenging to ionize. Our previous work established 2-(4-Boronobenzyl) isoquinolin-2-ium bromide (BBII) as a highly effective derivatization reagent for enhancing hydroxyl metabolite sensitivity in liquid chromatography-mass spectrometry. Building upon this, we herein introduce a novel and robust extension: BBII derivatization integrated with desorption electrospray ionization (DESI) for in situ hydroxyl metabolite detection. This innovative approach involves incorporating BBII directly into the DESI spray solvent, leading to significant sensitivity enhancement through instantaneous derivatization reaction. We demonstrate substantial signal increases ranging from 1.8- to 17.2-fold for hydroxyl metabolite reference standards, making previously undetectable compounds such as glucose, hexadecanol, and estradiol readily observable. When applied to mouse brain tissue sections for mass spectrometry imaging (MSI), BBII-DESI successfully revealed the distinct spatial distributions of representative hydroxyl metabolites, including glucose and cholesterol that are typically invisible to conventional methods. A key advantage of this methodology is the characteristic boron isotopic pattern of BBII-derivatized features, facilitating rapid and precise screening and identification. This BBII-assisted DESI strategy effectively unveils the "dark metabolome" of hydroxyl compounds, providing access to previously inaccessible metabolic information. Our method broadens the utility of ambient ionization techniques by enabling direct analysis of biological samples with minimal preparation, which is crucial for high-throughput applications. This advance offers substantial potential for accelerating biomarker discovery and disease diagnostics through direct visualization of metabolic alterations within native tissue environments, thereby marking a significant leap forward in spatial metabolomics applications for health and disease research.

DOI: https://doi.org/10.1021/acs.analchem.5c04858
Biomed Chromatogr. 2025 Dec;39(12): e70264

A Novel UPLC-MS/MS Method for Determining Tegoprazan in Rat Plasma: An Application in a Rat Pharmacokinetic Study.

Mengjie Xu, Yize Cai, Yu Wang, Manjie Lin, Qinghua Weng.

  Tegoprazan (TEG) is a commonly used drug in the treatment of gastroesophageal reflux disease. This work aimed to develop and validate an ultra-high-performance liquid chromatography-tandem mass Spectrometry (UPLC-MS/MS) method to determine the levels of TEG in rat plasma and to apply it for a rat pharmacokinetic study. Electrospray ionization source (ESI) positive and multiple reaction monitoring (MRM) mode were selected. The internal standard revaprazan (REV) and TEG were analyzed separately on a Waters ACQUITY UPLC BEH column. Gradient elution with a flow rate of 0.5 mL/min was used. Accuracy and precision were from -8.5% to 12.2%. Linearity was from 2 to 1000 ng/mL. IS-normalized recovery ranged from 109.3% to 113.6%. IS-normalized matrix effect was from 99.0% to 102.8%. The coefficient of variation of matrix effect was less than 10%. Dilution integrity showed that a tenfold dilution also met the guidelines. In the rat pharmacokinetic study, AUC(0 - ∞) and Cmax of TEG were 9129.6 ± 1823.3 μg/L*h and 2513.2 ± 707.0 ng/mL, respectively. Tmax and T1/2 were 1.8 ± 1.1 h and 1.6 ± 0.8 h, respectively. We finally established a rapid and robust UPLC-MS/MS quantitative analysis of TEG in rat plasma. Rat pharmacokinetics indicated that TEG was absorbed quickly and fast reached the maximum concentration.

Keywords:  UPLC–MS/MS; pharmacokinetics; rat; tegoprazan

DOI:  https://doi.org/10.1002/bmc.70264
J Chromatogr A. 2025 Nov 01. pii: S0021-9673(25)00852-0. [Epub ahead of print]1765 466508

Matrix effects in untargeted LC-MS metabolomics: From creation to compensation with post-column infusion of standards.

Pingping Zhu, Amy C Harms, Pascal Maas, Manisha Bakas, Julia Josette Whien, Anne-Charlotte Dubbelman, Thomas Hankemeier.

  Matrix effect is a well-known issue affecting accuracy and repeatability in metabolomics studies using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Post-column infusion of standards (PCIS) is a promising strategy to monitor and correct matrix effect but has been rarely reported in untargeted metabolomics. The major challenges lie in selecting appropriate PCISs and identifying the most suitable PCIS to correct the matrix effect experienced by each feature. In this study, we aim to present a method for selecting suitable PCISs for matrix effect compensation based on the artificial matrix effect (MEart) created by post-column infusion of compounds that disrupt the ESI process. Our hypothesis is that the suitable PCIS for a given analyte can be identified by comparing the PCISs' ability in MEart compensation. We evaluated this approach using 19 stable-isotopically labeled (SIL) standards spiked in plasma, urine, and feces. PCISs selected based on MEart were compared to those selected by biological matrix effect (MEbio), with 17 out of 19 SIL standards (89 %) showing consistent PCIS selection, demonstrating the effectiveness of MEart in identifying suitable PCISs. Applying MEart-selected PCISs to correct for the MEbio resulted in improved MEbio for most of the SILs affected by matrix effect and maintained MEbio for those experiencing no matrix effect. We demonstrated the efficacy of MEart in selecting suitable PCISs for MEbio correction within an LC-PCIS-MS method. Importantly, since MEart can be assessed for any detected feature, its application holds great potential for identifying suitable PCISs for matrix effect correction in untargeted metabolomics.

Keywords:  Artificial matrix; LC-MS; Matrix effect correction; Post-column infusion; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.chroma.2025.466508
J Chromatogr A. 2025 Nov 08. pii: S0021-9673(25)00869-6. [Epub ahead of print]1765 466525

Determination of residual 15 quaternary ammonium compounds in dairy products by dispersive solid-phase extraction purification with ultra-high performance liquid chromatography-tandem mass spectrometry.

Jiong Li, Jinhua Yan, Qingwen Cao, Dan Wu, Weijie Lin, Mengna Jin.

  The widespread use of quaternary ammonium compounds (QACs) could pose certain risks to human like potential food contamination. In this study, we developed a method to quantitate 15 QACs commonly found in milk, yogurt, and powdered milk via combining dispersive solid-phase extraction (DSPE) followed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Sample preparation methods and liquid chromatography conditions were established and optimized based on the inherent attributes of sample types and characteristics of the analyzed compounds to ensure measuring accuracy with minimal interference. Specifically, samples were extracted with a solvent of ACN/MeOH (8:2, v/v) supplemented with 0.2 % formic acid, followed by clean-up with a purification agent comprising NH2-PSA (200 mg) and SAX (100 mg). The mobile phase, consisting of a mixture of ACN/MeOH (4:6, v/v) and an aqueous solution, both with 0.2 % formic acid, facilitated the analysis of 15 targeted QACs on an Atlantis BEH C18 AX column using gradient elution. Subsequently, the QACs were ionized in ESI+ mode and quantitatively analyzed using MRM. With optimized parameters, the limits of detection (LODs) ranged from 0.05 to 0.50 μg/kg, with the limits of quantification (LOQs) ranging from 0.10 to 1.00 μg/kg. Additionally, the average recoveries ranged from 85.6 % to 100.7 %, while the precision for intra-day and inter-day ranged from 0.5 % to 7.4 % and 0.6 % to 8.7 %, respectively. Therefore, the method developed in this study was featured by its simplicity, accuracy, reliability, and suitability for determining QACs in milk, yogurt, and powdered milk.

Keywords:  Dairy product; Dispersive solid-phase extraction (DSPE); Quaternary ammonium compounds (QACs); Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)

DOI:  https://doi.org/10.1016/j.chroma.2025.466525
J Am Soc Mass Spectrom. 2025 Nov 14.

Sensitive and Selective Quantification of Acrylamide in French Fries and Canola Oil by Fluorous Derivatization with Liquid Chromatography-Tandem Mass Spectrometry.

Shimba Kawasue, Takahisa Shigematsu, Kento Sonogi, Reiko Koga, Tadashi Hayama, Hitoshi Nohta, Hideyuki Yoshida.

  Acrylamide is a carcinogen produced when foods containing sugar and asparagine are heated to 120 °C or higher during thermal cooking. As efforts are underway globally to reduce acrylamide intake, simple and practical methods to determine its levels in foods are needed. In this study, a highly sensitive and selective analytical method using liquid chromatography (LC) with tandem mass spectrometry (MS/MS) was developed for acrylamide detection. The α,β-unsaturated carbonyl structure in acrylamide was derivatized with perfluoroalkyl thiol, and the obtained derivative was analyzed based on fluorous properties without interference from food product contaminants. The feasibility of this method was demonstrated by analyzing common food products, such as French fries and canola oil. Acrylamide in the food samples was directly derivatized by adding a perfluoroalkyl thiol reagent-containing solution to the samples without any extraction steps. After delipidation, the fluorous-derivatized acrylamide was separated and detected using a fluorous LC column and an MS/MS system, respectively. The detection sensitivity of acrylamide using this method was 185-fold higher than that of the underivatized form. Therefore, this method is applicable for analyzing trace amounts of acrylamide in food samples and monitoring the acrylamide formation and migration processes during the cooking of French fries.

Keywords:  Acrylamide; Fluorous derivatization; Matrix effect; Tandem mass spectrometry

DOI:  https://doi.org/10.1021/jasms.5c00275
J Proteome Res. 2025 Nov 12.

Optimizing MS Parameters for Data-Independent Acquisition (DIA) to Enhance Untargeted Metabolomics.

Frederico G Pinto, Alexander D Giddey, Rouda S B Almarri, Omer S Alkhnbashi, Timothy J Garrett, Mohammed J Uddin, Nelson C Soares.

  Data-Independent Acquisition (DIA) has emerged as a powerful mass spectrometry (MS) strategy for comprehensive metabolomics. This study presents a novel short gradient (13 min) nanosensitive analytical method for human plasma analysis using DIA LC-MS/MS, focusing on in-depth optimization of MS parameters to maximize data quality and metabolite coverage. Key MS parameters, including scan speed, isolation window width, resolution, automatic gain control, and collision energy, were systematically tuned to balance the sensitivity and specificity while minimizing interferences. The optimized method enabled the detection of 2,907 features with 675 annotated compounds, leveraging recent progress in nano-LC-MS/MS for multiomics applications and showcasing the possibility of combining proteomics and metabolomics within a single chromatographic system. Ultimately, a comparison was performed between the data acquired through the DIA and DDA MS approaches in the context of untargeted metabolomics. This optimized analytical method yields more robust and reproducible results, thereby expanding the potential for meaningful discoveries across diverse biological fields.

Keywords:  data-independent acquisition; human plasma; mass spectrometry; metabolomics

DOI:  https://doi.org/10.1021/acs.jproteome.5c00622
J Pharm Biomed Anal. 2025 Nov 07. pii: S0731-7085(25)00561-8. [Epub ahead of print]269 117220

Simultaneous determination of 19 Aconitum alkaloids in whole blood and urine by ultra-high performance liquid chromatography-tandem mass spectrometry.

Zhaohong Wang, Meng Zhao, Yutong Wen, Jiao Wen.

  A rapid and sensitive UPLC-MS/MS method was developed and validated for the simultaneous quantification of nineteen Aconitum alkaloids in human blood and urine. Sample preparation utilized Oasis PRIME MCX μElution plates, which enabled efficient extraction and minimized matrix effects. The method demonstrated excellent selectivity, linearity (R² > 0.99), precision (RSDs < 9.1 %), and accuracy (±9 %). Stability testing revealed that benzoylated alkaloids exhibited notable degradation at room temperature within 8 h, likely due to hydrolysis； other alkaloids demonstrated varying degrees of stability under freeze-thaw cycles and long-term frozen storage. Extracted samples remained mostly stable when stored at 4°C for 24 h in the autosampler, emphasizing the importance of prompt analysis to ensure analyte integrity. Application to 25 fatal aconite poisoning cases revealed significant inter-individual variability, with generally higher alkaloid concentrations detected in urine, highlighting its utility as a sensitive matrix for exposure confirmation. A unique mass poisoning incident was characterized by the detection of only alcohol amine-type alkaloids without the highly toxic diester diterpenoid alkaloids, underscoring the need to expand analyte panels in toxicological investigations. Due to overlapping alkaloid profiles among Aconitum species and the impact of environmental and sample-handling factors, chemical profiling alone is insufficient for definitive species identification. Integrated multidisciplinary strategies are recommended for accurate diagnosis and forensic interpretation. This validated method provides a robust analytical platform to enhance forensic and clinical assessments of Aconitum poisoning.

Keywords:  Aconitum alkaloids; Alkaloids stability，Poisoning cases; Forensic toxicology; SPE; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2025.117220
Anal Bioanal Chem. 2025 Nov 11.

Rapid LC-MS/MS-based quantitative serum tryptophan metabolomics platform identifies and monitors novel biomarkers for diabetic kidney disease.

Lingshuai Zeng, Juan Bai, Xinrong Zou, Yi Zheng, Guangzhong Liu, Rui Lin, Yu Zhao, Xue Xue, Xiaoqin Wang.

  Diabetic kidney disease (DKD) is a leading cause of end-stage kidney disease worldwide. Conventional diagnostic tools lack sufficient sensitivity, necessitating novel biomarkers and reliable analytical platforms to improve DKD management. In this study, a serum tryptophan (TRP) metabolomics platform based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. After analytical optimization and validation, this platform was applied to a hospital-based case-control study (n = 200) for DKD. Associations between TRP metabolites and DKD risk were investigated via multivariable logistic regression. Metabolites demonstrating independent associations underwent diagnostic evaluation using receiver operating characteristic analysis with bootstrap internal validation. This LC-MS/MS platform enabled rapid quantification for TRP and 18 related metabolites within 6 min with low limits of detection, satisfactory recoveries, and acceptable matrix effects, meeting stringent analytical criteria suitable for clinical application. Seven TRP metabolites were significantly associated with DKD after covariate adjustment: 3-hydroxykynurenine (3-HK), 5-hydroxyindole acetic acid, indole-3-acetamide, and indole exhibited positive associations, while 3-hydroxyanthranilic acid, 5-hydroxytryptophan, and xanthurenic acid (XA) showed inverse associations. Individual metabolites demonstrated promising diagnostic performance (AUCs 0.762-0.896, sensitivities 65-80%, specificities 63-91%), except indole (AUC 0.683, sensitivity 51%, specificity 85%). Critically, the biomarker panel combining 3-HK and XA achieved exceptional diagnostic accuracy (AUC 0.970, sensitivity 92%, specificity 98%) for DKD. Bootstrap internal validation confirmed the stability of these findings. This study provided an LC-MS/MS-based platform for serum TRP metabolomics and identified a biomarker panel with exceptional diagnostic accuracy for DKD, offering significant potential as a clinically feasible tool for improving the diagnosis and management of DKD.

Keywords:  Biomarker; Diabetic kidney disease; Diagnosis; Liquid chromatography-tandem mass spectrometry; Metabolomics; Tryptophan metabolism

DOI:  https://doi.org/10.1007/s00216-025-06221-3
J Anal Toxicol. 2025 Nov 09. pii: bkaf100. [Epub ahead of print]

Stability of 22 Sedative-Type Drugs and Metabolites in Human Urine under Variable pH, Temperature, and Freeze-Thaw Conditions.

Feng-Shuo Yang, Shu-Huei Jian, Yi-Cheng Lee, Yung-Sheng Lan, Li-Ping Tseng, Yung-Hung Lee, Yi-Chen Chiu, Yi-Ching Lin.

  Ensuring analyte stability is essential for accurate forensic and clinical detection of sedative-type drugs. This study systematically evaluated the stability of 22 sedative-type drugs and metabolites in human urine under controlled conditions varying by pH (4.0, 7.0), temperature (25 °C, 4 °C, -20 °C), and freeze-thaw cycles (5 cycles), using a fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. While compounds such as midazolam, clobazam, and zolpidem remained highly stable, others-including alprazolam, triazolam, and lorazepam-exhibited notable degradation, particularly under acidic pH and elevated temperature. Flunitrazepam and clonazepam showed distinct degradation with the formation of 7-amino metabolites at neutral pH. Notably, this transformation occurred only in urine and not in phosphate-buffered saline, suggesting a urine-specific mechanism. These findings highlight the importance of compound-specific preservation strategies. In scenarios where analyte identity or sample pH cannot be verified promptly, immediate refrigeration or freezing (ideally at -20 °C), along with minimizing freeze-thaw cycles, is strongly recommended to preserve sample integrity and ensure reliable toxicological interpretation.

Keywords:  LC-MS/MS; analyte stability; benzodiazepines; urine; zolpidem

DOI:  https://doi.org/10.1093/jat/bkaf100
J Pharm Biomed Anal. 2025 Nov 09. pii: S0731-7085(25)00583-7. [Epub ahead of print]269 117242

Development of an LC-MS/MS method for the quantification of sunitinib and its metabolites in human nail and skin.

Miki Takenaka Sato, Takuya Araki, Hideaki Yashima, Jun Morita, Yoshiko Maeda, Masayuki Ohbayashi, Noriko Kohyama, Yoshio Ogawa, Takashi Fukagai, Koujirou Yamamoto, Mari Kogo.

  Sunitinib is associated with a high incidence of hand-foot skin reaction (HFSR) that significantly affects the quality of life and treatment continuation of the patient. The relationships between the concentrations of sunitinib and its metabolites in tissues and the occurrence of HFSR are crucial to preventing it. Therefore, this study developed a quantitative method for measuring the concentrations of sunitinib and its metabolites in human nails and skin. In addition, we evaluated whether this method can be applied to measure these drug concentrations in clinical samples. Nails or skin were crushed, methanol was added, and the supernatant obtained by centrifugation was used for analysis. Sunitinib, its metabolites, and voriconazole (internal standard: IS) were analyzed using liquid chromatography/mass spectrometry with electrospray ionization in positive mode. All samples were processed in the dark, and the analysis time was 5 min/run. In nail samples, calibration curves were linear in the range of 40-2000 pg/mg for sunitinib and N-desethyl sunitinib and 4-200 pg/mg for sunitinib N-oxide. In skin samples, linearity was observed in the range of 400-6000 pg/mg for sunitinib and N-desethyl sunitinib and 8-120 pg/mg for sunitinib N-oxide. The intra- and inter-day precision and accuracy were within 15 %, with a lower limit of quantification of 20 %. Extraction recoveries were ≥ 80 %, and the coefficients of variation for the IS-normalized matrix coefficients were < 15 %. This method was successfully applied to quantify these compounds in the nails and skin of patients receiving sunitinib treatment.

Keywords:  Human nail; Human skin; LC-MS/MS; N-desethyl sunitinib; Sunitinib; Sunitinib N-oxide

DOI:  https://doi.org/10.1016/j.jpba.2025.117242
Biomed Chromatogr. 2025 Dec;39(12): e70243

Quantitation of Plasma Bedaquiline Concentrations in Indian MDR-TB Patients Using Liquid Chromatography-Tandem Mass Spectrometry.

MDR‐TB MUKT and RePORT India Study Team

  Bedaquiline (BDQ) is a diarylquinoline used for the treatment of multidrug-resistant tuberculosis (MDR-TB) with a mechanism of action that inhibits the mycobacterial adenosine triphosphate synthase. There is a limited literature on the pharmacokinetics (PK) of BDQ in MDR-TB patients globally and even less from India. We aimed to develop a rapid, selective, sensitive, and robust high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to evaluate the PK of BDQ from the plasma of people with MDR-TB. The analyte was extracted from plasma by protein precipitation. The separation was achieved on a reverse phase C18 column with a mixture of aqueous and organic mobile phase in gradient mode at a flow rate of 0.3 mL/min and detected on a triple quadrupole mass spectrometer. Linearity was obtained between 0.0313 to 4.0 mg/L, and the method was validated following bioanalytical method validation guidelines. BDQ concentrations were determined from plasma samples, and intensive PK testing was performed in samples from 23 patients. The median Cmax attained was 1.59 mg/L with a Tmax around 6 h for most of our patients. This method was successfully developed and validated for a pilot PK study in Indian MDR-TB patients.

Keywords:  bedaquiline; liquid chromatography–tandem mass spectrometry; multidrug‐resistant tuberculosis; pharmacokinetics; plasma

DOI:  https://doi.org/10.1002/bmc.70243
J Clin Med. 2025 Oct 24. pii: 7544. [Epub ahead of print]14(21):

UHPLC-MS/MS for Antipsychotic Drug Monitoring: A Systematic Review of Clinical and Analytical Performance.

Ciprian-Ionuț Băcilă, Bianca-Maria Macavei, Monica Cornea, Bogdan Ioan Vintilă, Andrei Lomnășan, Claudia Elena Anghel, Andreea Maria Grama, Cristina Elena Dobre, Claudia Marina Ichim, Gabriela Cioca.

  Background/Objectives: Therapeutic drug monitoring (TDM) of antipsychotic medications plays an important role in optimizing treatment efficacy, reducing adverse effects, and supporting adherence. While Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) has long been the gold standard for antipsychotic quantification, recent advances in automated platforms and microsampling raise questions about its current clinical practicality. This systematic review evaluated the clinical applicability and analytical performance of UHPLC-based methods for monitoring antipsychotic drugs, focusing on precision, recovery, matrix effects, and suitability across various biological matrices. Methods: A systematic search of PubMed, Scopus, and Web of Science was conducted for studies published between 2013 and 2024 involving UHPLC-based quantification of antipsychotics in clinical samples from adult patients. Data on analytical parameters, sample matrices, and study characteristics were extracted. A custom quality checklist was used to assess methodological rigor. In addition to qualitative synthesis, non-traditional quantitative approaches were applied, including descriptive aggregation of recovery, matrix effects, and precision across studies, as well as correlation analyses to explore relationships among performance parameters. Results: Twelve studies were included, spanning a range of typical and atypical antipsychotics and metabolites. Plasma and serum demonstrated the highest analytical reliability (recovery >90%, minimal matrix effects), while dried blood spots (DBSs), whole blood, and oral fluid showed greater variability. Clinically, UHPLC-MS/MS enabled more accurate dose adjustments and identification of non-adherence, outperforming immunoassays in sensitivity, specificity, and metabolite detection. Microsampling methods showed promise for outpatient and decentralized care but require further clinical validation. Conclusions: UHPLC-MS/MS remains the most robust and reliable method for TDM of antipsychotics, especially when quantification of active metabolites is required. While logistical barriers remain, technological advances may enhance feasibility and support broader integration into routine psychiatric care.

Keywords:  TDM; UHPLC; antipsychotics; clinical psychiatry

DOI:  https://doi.org/10.3390/jcm14217544
J Chromatogr A. 2025 Nov 04. pii: S0021-9673(25)00862-3. [Epub ahead of print]1765 466518

Development of an effective method for biomonitoring of per- and polyfluoroalkyl substances and phthalate diesters and monoesters in human hair using liquid chromatography coupled with tandem mass spectrometry.

Huiyang Wang, Guanxi Ye, Qihui Zhang, Zongrui Li, Hui Yang, Ying Zhou, Senhao Xu, Jingxin Wang, Yunjiang Yu.

  Human hair serves as a validated non-invasive biomonitoring tool for evaluating human exposure to specific emerging pollutants. In the present study, an effective and reliable method for the simultaneous determination of 24 phthalate diesters (PAEs) and monoesters (mPAEs) as well as 36 per- and polyfluoroalkyl substances (PFASs) has been developed and validated in human hair based on ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-ESI-MS/MS). Sample preparation protocols include washing and grinding of hair, ultrasonic solvent extraction, and dispersive solid-phase extraction (d-SPE) cleanup. The proposed method was verified through blank and matrix spiking analysis. The recoveries of the analytes ranged from77 % to 117 % for PAEs and mPAEs with relative standard deviations (RSDs) below 20 %, and from 83 % to 117 % for PFASs with RSDs below 20 %. All the internal standards were recovered between 82±2 % to 105±5 %. The limits of detection (LODs) of PAEs and mPAEs were from 0.042 to 24 ng/g and from 0.002 to 1.25 ng/g for PFASs. The developed method was applied to measure target analytes in actual hair samples which exhibited that DEHP and MEHP emerged as the predominant PAEs and mPAEs compounds while PFOA and PFOS were the most abundant PFASs. The developed method indicated robust performance in hair analysis of PFASs and PAEs for biomonitoring.

Keywords:  Biomonitoring; Emerging contaminants; Hair analysis; Method development; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2025.466518
J Pharm Biomed Anal. 2025 Nov 06. pii: S0731-7085(25)00565-5. [Epub ahead of print]269 117224

Automated magnetic bead‑based LC-Triple Quadrupole MS approach for sensitive colistin therapeutic drug monitoring in human plasma.

Fangqiong Li, Juan Wang, Xiaoting Gong, Ji Shen, Qiuyu Zhu, Chuanwei Xin, Wei Zhang, Haiying Wu, Huifang Jiang, Yuexing Tu.

  Colistin, a cationic peptide antibiotic produced by Bacillus polymyxa subsp. colistinus, is a last-line defense against multidrug-resistant Gram-negative bacteria. However, its clinical utility is hampered by significant nephrotoxicity and neurotoxicity at supratherapeutic levels and the risk of promoting antimicrobial resistance (AMR) at subtherapeutic concentrations. To enable precise therapeutic drug monitoring (TDM) and mitigate these risks, we developed a fully automated magnetic bead-based LC-Triple Quadrupole MS method for sensitive quantification of colistin in human plasma. The method utilizes hydrophilic-lipophilic balance (HLB) magnetic beads coupled with an automated 96-well extraction system. Colistin A, colistin B, and the internal standard (polymyxin B1) were analyzed via LC-Triple Quadrupole MS employing positive electrospray ionization. Method validation was performed in accordance with FDA guidance. The method exhibited excellent linearity, sensitivity, precision, accuracy, recovery, and minimal matrix effects. The assay demonstrated the broadest quantifiable range reported to date (0.01-20 µg/mL) for colistin A and B in a 5.0-min runtime. The lower limit of quantification (LLOQ) was 0.01 µg/mL, ensuring high sensitivity and specificity with negligible endogenous interference. This automated, cost-effective, and robust platform facilitates reliable colistin TDM, supporting personalized dosing strategies to optimize efficacy and minimize toxicity.

Keywords:  Antibiotic quantification; Colistin; Hydrophilic-lipophilic balance magnetic beads; Liquid chromatography–triple quadrupole mass spectrometry; Plasma extraction; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jpba.2025.117224
Foods. 2025 Oct 24. pii: 3636. [Epub ahead of print]14(21):

Development and Validation of a QuEChERS-Based LC-MS/MS Method for Natamycin in Imported Agricultural Commodities in Korea.

Ga-Eul-Hae An, Joon-Kyung Oh, Jae-Hyeong Kim, Hee-Ra Chang.

  Natamycin is widely used in other countries for the postharvest treatment of agricultural commodities to prevent fungal growth. However, since no MRL has been set in Korea, natamycin residues are regulated under the Positive List System (PLS) with a uniform limit of 0.01 mg/kg, requiring the development of highly sensitive and reliable analytical methods. In this study, a QuEChERS-based analytical method was developed and validated for the quantification of natamycin in five agricultural commodities-soybean, mandarin, hulled rice, green pepper, and potato-using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extraction using methanol with 3 g of MgSO4 resulted in high recoveries without crystallization, and clean-up with MgSO4 and C18 effectively reduced matrix interferences blow <50%. Natamycin was detected in all five matrices at 6.8 min without any interfering peaks. The MLOQ was determined at 0.01 mg/kg for all five matrices. The mean recoveries (82.2-115.4%) and %CV values (1.1-4.6%) values were within the acceptance criteria defined by the CODEX guidelines. Matrix effects were classified as "soft" for mandarin (|ME| < 20%) and "medium" for soybean, hulled rice, green pepper, and potato (20% ≤ |ME| < 50%). The analytical method for natamycin was validated as suitable for regulatory safety monitoring under the Korean PLS.

Keywords:  PLS; food safety; matrix effects; pesticides; residues

DOI:  https://doi.org/10.3390/foods14213636
Forensic Sci Med Pathol. 2025 Nov 10.

A systematic review of organophosphorus pesticides detection in biological matrices.

Shanu Kumar, Bhoopendra Singh, Sraddha Singh, Hritik Kumar.

   PURPOSE: Organophosphorus pesticides (OPPs) are widely used for pest control; however, their accessibility has led to significant misuse, contributing to self-poisoning, morbidity, and mortality, particularly in low- and middle-income countries. Accurate detection of OPPs in biological samples is critical for forensic investigations of poisoning cases. This systematic review evaluates recent advancements in extraction and analytical methods for the detection of OPPs in biological matrices, focusing on diagnostic accuracy and forensic applicability.
MATERIALS AND METHODS: A comprehensive literature search was conducted in PubMed, Scopus, Cochrane Library, and Google Scholar for studies published between January 2014 and July 2024. Predefined search terms were used in various Boolean combinations. Studies were screened according to inclusion and exclusion criteria based on the Population, Intervention, Comparison, and Outcome (PICO) framework. Methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2), and the risk of bias was evaluated using the Cochrane diagnostic test accuracy tool.
RESULTS: Twenty-one eligible studies were included, reporting a variety of extraction and detection methods for OPPs across multiple biological matrices such as blood, urine, hair, and viscera. Techniques including Gas chromatography-tandem- tandem mass spectrometry (GC-MS/MS), Liquid chromatography-tandem mass spectrometry (LC-MS/MS), High-performance liquid chromatography (HPLC), and High-performance thin-layer chromatography (HPTLC) demonstrated high sensitivity and specificity. Innovations in extraction, such as solid-phase microextraction (SPME) and microwave-assisted extraction, provided rapid, cost-effective, and environmentally friendly alternatives to conventional methods.
CONCLUSIONS: Significant progress has improved OPP detection in forensic toxicology, though challenges like matrix effects, lab standardization, and broader applicability persist. Continued research and method optimization are essential to improve reliability and routine forensic implementation.

Keywords:  Analytical techniques; Biological matrices; Extraction methods; Forensic toxicology; Organophosphorus pesticides; Poisoning

DOI:  https://doi.org/10.1007/s12024-025-01094-5
J Vis Exp. 2025 Oct 24.

Sequential Detection of Biomolecules in Formalin-fixed, Paraffin-embedded Samples with Mass Spectrometry Imaging.

Erin H Seeley, Danielle L Stolley, Anna K Casasent, Basant T Gamal, Sammy Ferri-Borgogno, Jared K Burks.

Tissues are complex cellular environments, made up of a vast array of cell types and biomolecules all interacting with each other to carry out the functions of the organ. Traditionally, techniques for the analysis of biomolecules such as metabolites, glycans, and proteins involved the homogenization of tissue, destroying all spatial information. These traditional methods inhibited the complete understanding of complex intra- and extracellular molecular interactions. Mass Spectrometry Imaging (MSI), on the other hand, not only preserves the spatial information of biomolecules in tissue but also allows for multiple classes of analytes to be detected from the same tissue section through sequential analysis at a near-single-cell resolution. This enables us to derive a more complete picture of molecular interactions across different classes of biomolecules. The protocol presented here outlines the steps for performing mass spectrometry imaging of metabolites, N-linked glycans, and tryptic peptides sequentially from the same tissue section at a 20 µm resolution. Conscientious consideration of the order in which the classes of analytes are imaged, along with careful handling of the sections to ensure integrity, allows for multiple high-quality images to be collected from the same section. These data can subsequently be integrated with other spatial omics data (transcriptomics, immunohistochemistry, etc.) collected from serial sections, where the same cell can be analyzed in these adjacent sections.

DOI: https://doi.org/10.3791/68618
Anal Chem. 2025 Nov 12.

Integrated Analytical System for Comprehensive Two-Dimensional Enantio-Gas Chromatography and Low-Pressure Gas Chromatography Mass Spectrometry Utilizing a Switching Valve: Design and Optimization.

Giorgia Rinaldi, Antonio Ferracane, Mariosimone Zoccali, Luigi Mondello.

An analytical system was developed to perform both comprehensive two-dimensional enantio-gas chromatography mass spectrometry with the second-dimension column working under low-pressure conditions (eGC×LP-GC-QMS) and low-pressure gas chromatography mass spectrometry (LP-GC-QMS) analysis, without any changes to the instrumental setup. This is achieved using a switching valve, which also allows for back-flushing of the first-dimension column, thereby generating a flexible and powerful analytical platform. The eGC×LP-GC-QMS configuration can provide both targeted and untargeted information in a single run. In this research, it is employed for the analysis of 56 pesticides (without considering isomers), with the added capability of the backflush mode, effectively removing high-boiling compounds extracted from the matrix and improving sample throughput and system robustness, without major concerns regarding sample preparation. The first-dimension is a chiral column, which enables the separation of seven enantiomers: acephate, benoxacor, chlorflurecol, dibrom, fipronil, methamidophos, and propetamphos. The second-dimension is a wide-bore column (5 m × 0.53 mm I.D. × 0.53 μm df) operating under vacuum conditions. Thanks to the use of a switching valve, it is possible to directly perform LP-GC-QMS analysis, enabling the investigation of a broader range of compounds in terms of boiling point. The operability range of volatile analytes was evaluated for both configurations by injecting different alkane mixtures: C7-C30 for the eGC×LP-GC-QMS configuration and C7-C40 for the LP-GC-QMS configuration.

DOI: https://doi.org/10.1021/acs.analchem.5c05852
Food Res Int. 2025 Dec;pii: S0963-9969(25)01823-X. [Epub ahead of print]221(Pt 3): 117485

Applications of metabolomic techniques to explore the diversity of phenolic compounds in pseudocereals: Effects of processing and health implications.

Ishika Jain, Deepika Kathuria, Maman Paul, Narpinder Singh.

  Pseudocereals such as amaranth, buckwheat, and quinoa are gaining attention for their exceptional nutritional profile and health-promoting properties. Unlike traditional cereals, pseudocereals are rich in phenolic compounds, which have been characterized using metabolomics approaches, enhancing their value for food security and combating micronutrient deficiencies. Metabolomics, a powerful analytical tool, has been instrumental in characterizing the diverse bioactive compounds in these grains, with a focus on phenolic compounds that contribute to their antioxidant, anti-inflammatory, antidiabetic, and anticancer properties. This review summarizes recent advancements in metabolomics approaches, including liquid chromatography-mass spectrometry (LC-MS) and ultra-high-performance liquid chromatography (UHPLC), to identify and quantify the phenolic composition of pseudocereals. It also discusses the impact of various environmental factors, genetic diversity, and food processing techniques, such as fermentation, roasting, and germination, on the metabolic profile of these grains.

Keywords:  Amaranth; Buckwheat; Metabolomics; Phenolic compounds; Pseudocereals; Quinoa

DOI:  https://doi.org/10.1016/j.foodres.2025.117485