bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–08–03
sixty-one papers selected by
Sofia Costa, Matterworks



  1. J Mass Spectrom. 2025 Aug;60(8): e5159
      Bioanalytical method development and validation are essential for reliable quantification of drugs in biological matrices. This research focuses on developing and validating a UPLC-MS/MS method for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma, adhering to established regulatory guidelines for bioanalytical method validation. The development and validation focus on creating a robust and sensitive assay suitable for bioequivalence studies and routine therapeutic drug monitoring. This method utilizes bisoprolol D5 and hydrochlorothiazide C13 D2 as internal standards to enhance accuracy and precision. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm particle size) with an isocratic mobile phase consisting of 10 mM ammonium formate buffer, methanol, and 0.1% ammonia solution (10:90, v/v). The flow rate was set at 0.3 mL/min, with a retention time of 2.2 min. Multiple reaction monitoring (MRM) was used with positive ESI for bisoprolol (m/z 326.36 → 116.13, internal standard m/z 331.36 → 121.13) and negative ESI for hydrochlorothiazide (m/z 296.11 → 269.00, internal standard m/z 299.11 → 270.00). The analytes and their respective internal standards were co-extracted using a liquid-liquid extraction method with tert-butyl methyl ether as the extraction solvent. Linearity for bisoprolol and hydrochlorothiazide was maintained over a concentration range of 1-100 ng/mL for bisoprolol and 1.0-300 ng/mL for hydrochlorothiazide, respectively, using a weighted least squares linear regression model (1/x). This method achieved a lower limit of quantification (LLOQ) of 1.0 ng/mL, making it highly sensitive for the detection of these analytes. Moreover, the method demonstrated high accuracy, precision, selectivity, and reduced overall analysis time, making it well suited for routine analysis and bioequivalence studies of 10 mg bisoprolol and 25 mg hydrochlorothiazide tablets.
    Keywords:  UPLC‐MS/MS; bisoprolol; hydrochlorothiazide; liquid–liquid extraction; method development; method validation
    DOI:  https://doi.org/10.1002/jms.5159
  2. Anal Chim Acta. 2025 Oct 08. pii: S0003-2670(25)00748-2. [Epub ahead of print]1370 344354
      The accurate determination of amino compounds in complex matrices is essential for a wide range of applications, from bioanalysis to industry. Liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful analytical technique for their identification and quantification. However, the low ionization efficiency and poor retention of some amino compounds present a challenge in mass spectrometric detection, making derivatization a crucial step. This tutorial review provides a comprehensive guide to improving LC-MS methods through derivatization, enhancing detection and quantification. Key aspects of the optimization method are covered, including selection of suitable derivatization reagents, optimization of reaction and chromatographic conditions, and other practical aspects. Drawing on an extensive literature review and experience, this tutorial offers researchers valuable insights into refining LC-MS workflows for methods using derivatization, mainly to determine amino acids and biogenic amines. Serving as both an instructional guide and a practical reference, this study aims to advance the effectiveness of LC-MS-based methods for amino compound determination.
    Keywords:  Amino acids; Derivatization; Liquid chromatography; Mass spectrometry
    DOI:  https://doi.org/10.1016/j.aca.2025.344354
  3. Turk J Pharm Sci. 2025 Aug 01. 22(3): 191-206
       Objectives: A novel, high-throughput liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technique has been developed that uses Etravirine (ETR) as the internal standard (IS) to simultaneously quantify Doravirine (DOR), Lamivudine (LAM), and Tenofovir Disoproxil Fumarate (TDF) in human plasma. The procedure employs a precipitation extraction technique to analyze analytes from human plasma. This study aims to develop and validate a novel and reliable stability-indicating UPLC-MS/MS method for the simultaneous determination of DOR, LAM, and TDF in human plasma, using ETR as the IS.
    Materials and Methods: ETR, based on its stable-isotopic nature and structural and physicochemical similarity to the analytes of interest, was used as an IS. Precipitation extraction was the technique used to prepare samples. An agilent zorbax XDB C18 analytical column (2.1 × 50 mm, 3.5 μm) was used for chromatographic separation, and its isocratic mobile phase consists of acetonitrile and buffer (5 mM of ammonium formate with 0.1 % formic acid) in the ratio 80:20, v/v, at a flow rate of 0.120 mL/min.
    Results: The parent-to-product ion transitions for the drugs were as follows : LAM: m/z 231.08 amu → 112.00 amu, TDF: m/z 288.33 amu → 176.17 amu, DOR: m/z 426.38 amu → 112.02 amu, and IS ETR: m/z 437.36 amu → 164.97 amu. These transitions were observed using a triple quadrupole mass spectrometer in the multiple reaction monitoring (MRM) positive ion mode. The compound's basic group content determined which positive mode to choose. For DOR, LAM and TDF, the method was validated throughout concentration ranges of 2.5-1000 ng/mL with correlation coefficients (r2) values obtained were found to be 0.99. From spiked plasma samples, the mean recovery outcomes were observed and found to be DOR, LAM, and TDF was 83.39%, 87.33%, and 85.56%. With a 3.0-minute total run time, the approach was shown to be reliable and quick.
    Conclusion: A triple quadrupole mass spectrometer running in the MRM positive ion mode was used to track these transitions. The compounds' functional group content served as the basis for choosing the positive mode. The mean recovery values were obtained for three APIs from spiked plasma samples. The run times were found to be both reliable and quick. The method was proven to produce precise and specific results for the determination of selected drugs through the current study. The method is stable when exposed to various stress conditions, demonstrating minimal degradation. The current method was validated as per the ICH M10 guidelines and was found to meet the desired acceptance criteria. The developed bioanalytical method, validated in accordance with ICH M10 guidelines, demonstrated high accuracy, precision, and reproducibility for the simultaneous quantification of DOR, LAM, and TDF. Its streamlined design and reliable performance make it a valuable tool for routine analysis.
    Keywords:  Doravirine; Tenofovir Disoproxil Fumarate; UPLC-MS/MS; lamivudine; quantification
    DOI:  https://doi.org/10.4274/tjps.galenos.2025.70718
  4. J Pharm Biomed Anal. 2025 Jul 16. pii: S0731-7085(25)00409-1. [Epub ahead of print]266 117068
      Optimizing run time in gas chromatography-mass spectrometry (GC-MS) based metabolomics is essential for balancing metabolite coverage, reproducibility, and practical workflow constraints. In this study, three GC-MS methods with different run times, short (26.7 min), a standard method based on the established Fiehn protocol (37.5 min), and long (60 min), were evaluated across three biological matrices: cell culture, plasma, and urine. All methods were applied using identical injection volumes and derivatization protocols. The number of annotated metabolites in the short and standard methods was comparable: 138 vs. 156 in cell culture, 147 vs. 168 in plasma, and 186 vs. 198 in urine. The long method provided higher metabolite coverage (196 in cell culture, 175 in plasma, 244 in urine), largely due to improved chromatographic resolution and deconvolution, which also increased the number of unannotated features. Although the proportion of high-filling (0.75-1) annotated metabolites was similar across all methods (∼79-90 %), repeatability was slightly better in the standard and long methods (RSD ∼20-24 %) than in the short method (RSD ∼23-30 %). Notably, since derivatized samples must be analyzed within 24 h, the short method presents a practical advantage by enabling completion of full batch analysis within this time constraint. Overall, while the short and standard methods offer similar identification performance, the long method enhances analytical depth.
    Keywords:  Cell culture; Data analysis; GC-MS; Metabolomics; Plasma; Untargeted; Urine
    DOI:  https://doi.org/10.1016/j.jpba.2025.117068
  5. Molecules. 2025 Jul 16. pii: 2995. [Epub ahead of print]30(14):
      Sitagliptin is an orally bioavailable selective DPP4 inhibitor that reduces blood glucose levels without significant increases in hypoglycemia. The aim of this study was to design and validate an innovative, rapid, and highly sensitive LC-MS/MS assay for the precise measurement of sitagliptin concentrations in human plasma. This analytical method, utilizing sitagliptin-d4 as the internal standard, is performed using only 100 μL of plasma and a liquid-liquid extraction procedure based on methyl tert-butyl ether (MTBE). Chromatographic separation is expertly achieved with a Kinetex® C18 column under isocratic elution, employing a perfect 1:1 blend of 5 mM ammonium acetate (with 0.04% formic acid) and acetonitrile, and maintaining an efficient flow rate of 0.2 mL/min. Detection occurs in positive ionization mode through multiple reaction monitoring, precisely targeting transitions of m/z 408.2 → 193.0 for sitagliptin and 412.2 → 239.1 for the IS. The total runtime of this assay is under 2 min. Comprehensive validation in line with MFDS and FDA criteria demonstrates outstanding linearity (5-1000 ng/mL, r2 > 0.998), alongside impressive levels of accuracy, precision, recovery and sample stability. Due to its minimal sample requirement and high-throughput capability, the validated approach is highly appropriate for pharmacokinetic and bioequivalence assessments involving sitagliptin.
    Keywords:  LC-MS/MS; bioanalytical method validation; dipeptidyl peptidase-4 inhibitor; human plasma; pharmacokinetics; sitagliptin
    DOI:  https://doi.org/10.3390/molecules30142995
  6. Nat Commun. 2025 Jul 26. 16(1): 6911
      Metabolite identification in non-targeted mass spectrometry-based metabolomics remains a major challenge due to limited spectral library coverage and difficulties in predicting metabolite fragmentation patterns. Here, we introduce Multiplexed Chemical Metabolomics (MCheM), which employs orthogonal post-column derivatization reactions integrated into a unified mass spectrometry data framework. MCheM generates orthogonal structural information that substantially improves metabolite annotation through in silico spectrum matching and open-modification searches, offering a powerful new toolbox for the structure elucidation of unknown metabolites at scale.
    DOI:  https://doi.org/10.1038/s41467-025-61240-z
  7. Sci Rep. 2025 Jul 29. 15(1): 27728
      Omadacycline, a third-generation tetracycline antibiotic, exhibits time-dependent pharmacokinetics. The ratio of the 24-hour area under the concentration-time curve to minimum inhibitory concentration (AUC0-24/MIC) serves as the primary pharmacokinetic/pharmacodynamic (PK/PD) index for omadacycline, demonstrating strong correlation with therapeutic efficacy, particularly in critically ill patient populations. To facilitate routine therapeutic drug monitoring (TDM) in clinical practice, a high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of omadacycline in human plasma. An Agilent 1260 series liquid chromatograph and an API 4000 triple tandem quadrupole mass spectrometer were used for the determination of omadacycline and endocannabinoids. The separation was performed on a Phenomenex KINETEX XB-C18 column (2.6 μm, 3 × 50 mm) with 0.1% formic acid-water and pure acetonitrile as the mobile phases. The LC-MS/MS separation was carried out using a gradient elution procedure at a flow rate of 0.4 mL/ min, and the total run time was 5 min. An electrospray ionization (ESI) was selected, and the detection ions of omadacycline and the internal standard (fexofenadine-d6) were determined by mass spectrometry scanning with multiple reaction monitoring (MRM) in positive ion mode as follows: m/z 557.4→453.4 and m/z 508.4→472.8, respectively. The established linear range (20-2000 ng/mL) effectively covers the plasma concentration range encountered in > 98% of clinical samples, making it suitable for routine therapeutic drug monitoring. The method demonstrated acceptable selectivity, recovery, and matrix effect. The results of intra-day precision and inter-day precision show that the relative standard deviation (RSD) is less than 10%, while the relative error (RE) is within ± 10.00%. In this study, we developed and validated a simple, sensitive and accurate method to quantify the concentration of omadacycline in human plasma using LC-MS/MS. This offers essential technical support for the rational clinical application of omadacycline.
    Keywords:  LC-MS; Omadacycline; Plasma; Therapeutic drug monitoring
    DOI:  https://doi.org/10.1038/s41598-025-13396-3
  8. J Pharm Biomed Anal. 2025 Jul 23. pii: S0731-7085(25)00419-4. [Epub ahead of print]266 117078
      A reversed-phase liquid chromatography-tandem mass spectrometry method was developed and validated for quantifying nine novel oral targeted anticancer agents mainly indicated for non-small cell lung cancer: adagrasib, capmatinib, ensartinib, entrectinib, larotrectinib, lorlatinib, pralsetinib, selpercatinib and sotorasib in human plasma for therapeutic drug monitoring. Chromatographic separation used an Acquity BEH C18 column with step gradient of 0.1 % formic acid in water and acetonitrile-methanol (50:50, v/v), at a 0.5 mL/min flow rate. Plasma samples were pretreated via precipitation with acetonitrile and diluted in 0.1 % formic acid in water. The reversed-phase chromatography was coupled with tandem mass spectrometry in positive ion mode. The assay was successfully validated over the following ranges: 100 - 10,000 ng/mL for adagrasib, capmatinib, entrectinib, pralsetinib, selpercatinib; 50 - 50,000 ng/mL for ensartinib; 10 - 1000 ng/mL for larotrectinib, lorlatinib; and 10 - 10,000 ng/mL for sotorasib. Accuracy and precision met the predefined criteria. Stability tests confirmed that all analytes were stable in plasma for up to 157 days at -20°C except for entrectinib, which was stable for 35 days at -20°C. At room temperature, the analytes were at least stable in plasma for 7 days, however, for adagrasib, entrectinib and sotorasib, stability for up to 3 days could be demonstrated. We recommend sending these samples on dry ice or refrigerated. After the validation, 74 plasma samples were measured in the application phase and all results but one fell within the validated ranges. This assay allows simultaneous quantification of nine novel targeted therapies and supports therapeutic drug monitoring.
    Keywords:  Bioanalysis; LC-MS/MS; Non-small cell lung cancer; Targeted therapy; Therapeutic Drug Monitoring (TDM)
    DOI:  https://doi.org/10.1016/j.jpba.2025.117078
  9. Biomed Chromatogr. 2025 Sep;39(9): e70179
      Measuring cobicistat and venetoclax concentrations in human plasma and serum facilitates therapeutic drug monitoring (TDM) and pharmacokinetic (PK) boosting studies. Therefore, the objective of this study was to develop and validate a rapid LC-MS/MS analytical method for the simultaneous determination of cobicistat and venetoclax concentrations in plasma and serum. The method was validated according to EMA and FDA guidelines. Chromatographic separation was performed using a liquid chromatography (LC) system with a C18 column. The elution gradient involved two mobile phases: mobile phase A (ammonium formate) and mobile phase B (acetonitrile). The concentration range was 5-500 μg/L for cobicistat and 50-5000 μg/L for venetoclax. Accuracy and precision were within the required limits, with accuracy ranging from -5.9% to 2.4%, within-day precision from 1.2% to 4.8%, and between-day precision from 0.4% to 4.3%. Cobicistat and venetoclax were stable for at least 8 days under various storage and handling conditions. Clinical TDM samples showed mean concentrations ± standard deviation (SD) of 138.8 ± 123.3 μg/L for cobicistat and 1497.1 ± 1285.9 μg/L for venetoclax. The development and validation of this LC-MS/MS assay provide a reliable and efficient method for the simultaneous quantification of cobicistat and venetoclax in plasma and serum samples.
    DOI:  https://doi.org/10.1002/bmc.70179
  10. J Am Soc Mass Spectrom. 2025 Jul 25.
      Analyzing metabolites using mass spectrometry provides valuable insight into an individual's health or disease status. However, various sources of experimental variation can be introduced during sample handling, preparation, and measurement, which can negatively affect the data. Quality assurance and quality control practices are essential to ensuring accurate and reproducible metabolomics data. These practices include measuring reference samples to monitor instrument stability, blank samples to evaluate the background signal, and strategies to correct for changes in instrumental performance. In this context, we introduce mzQuality, a user-friendly, open-source R-Shiny app designed to assess and correct technical variations in mass spectrometry-based metabolomics data. It processes peak-integrated data independently of vendor software and provides essential quality control features, including batch correction, outlier detection, and background signal assessment, and it visualizes trends in signal or retention time. We demonstrate its functionality using a data set of 419 samples measured across six batches, including quality control samples. mzQuality visualizes data through sample plots, PCA plots, and violin plots, which illustrate its ability to reduce the effect of experiment variation. Compound quality is further assessed by evaluating the relative standard deviation of quality control samples and the background signal from blank samples. Based on these quality metrics, compounds are classified into confidence levels. mzQuality provides an accessible solution to improve the data quality without requiring prior programming skills. Its customizable settings integrate seamlessly into research workflows, enhancing the accuracy and reproducibility of the metabolomics data. Additionally, with an R-compatible output, the data are ready for statistical analysis and biological interpretation.
    Keywords:  Data quality; Mass spectrometry; Metabolomics; R package; R-shiny app
    DOI:  https://doi.org/10.1021/jasms.5c00073
  11. J Mass Spectrom Adv Clin Lab. 2025 Aug;37 56-64
       Objective: Tandem mass spectrometry (MS/MS) is highly specific in principle, but there is always the possibility of interference due to unexpected substances in the samples that have the identical mass transitions as the target analytes (isomeric/isobaric interferences). By recording the ion ratio (IR), clinical laboratories already widely attempt to identify such interferences in individual cases. To supplement this procedure, differential tuning effects can be assessed. We aimed to evaluate this approach experimentally.
    Methods: The detuning ratio (DR) is based on the differential influences of MS instrument settings on the ion yield of a respective target analyte; isomeric or isobaric interferences can lead to a shift of the DR for an affected sample. By determining the DR in samples in which known isomeric interference substances have been spiked to the target analyte, the applicability of DR detection was quantitatively investigated.
    Results: It was observed in two independent exemplary test systems (Cortisone / Prednisolone and O-Desmethylvenlafaxine / cis-Tramadol HCl) that a DR can indicate the presence of isomeric interferences.
    Conclusion: It was confirmed that a DR can be used as a method to obtain indications of the presence of isomeric or isobaric interferences in individual samples in an analytical LC-MS/MS system; the technique can be used in addition to the established method of IR detection to increase the analytical reliability of clinical MS analyses.Abbreviations: CE, collision energy; CID, collision induced dissociation; CXP, cell exit potential; CLSI, Clinical and Laboratory Standards Institute; DR, detuning ratio; ESI+, positive electrospray ionization; IR, ion ratio; IS, internal standard; LC, liquid chromatographic; LC-MS/MS, liquid chromatography tandem mass spectrometry; ME, matrix effects; MRM, multiple reaction monitoring; MS, mass spectrometry; m/z, mass-to-charge ratio; TIC, Total ion current.
    Keywords:  Detuning ratio; Interference; Ion ratio; LC-MS/MS; Tandem-mass spectrometry
    DOI:  https://doi.org/10.1016/j.jmsacl.2025.07.002
  12. Rapid Commun Mass Spectrom. 2025 Nov 15. 39(21): e10117
       RATIONAL: Investigating TCE glutathione (GSH) conjugation metabolites is essential, as these reactive intermediates play a central role in TCE bioactivation and are implicated in organ-specific toxicities, including nephrotoxicity, hepatotoxicity, and neurotoxicity. Characterizing and quantifying these metabolites enhances our understanding of TCE metabolism, supports biomarker discovery, and helps elucidate mechanisms of TCE-induced toxicity.
    METHODS: A robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the detection and quantification of three major GSH-related TCE metabolites: S-(1,2-dichlorovinyl)-glutathione (DCVG), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAcDCVC). Serum samples were obtained from male C57BL/6 mice chronically exposed to TCE (100 ppm in drinking water for 15 weeks).
    RESULTS: Calibration curves for all three metabolites demonstrated excellent linearity (R2 > 0.998). The method achieved limits of detection (LOD) ranging from 0.0057 to 0.0120 nM, limits of quantitation (LOQ) from 0.0189 to 0.0401 nM, recoveries of 75.9%-115.5%, and inter-assay variation of 0.5%-11.5%. PD model mice exhibited elevated serum levels of DCVG and DCVC, while NAcDCVC levels were significantly reduced.
    CONCLUSION: This study presents the first comprehensive LC-MS/MS-based quantification of TCE GSH conjugation metabolites in serum, offering high sensitivity, precision, and reproducibility. The observed elevated serum levels of the toxic metabolites DCVG and DCVC, along with the markedly reduced NAcDCVC concentrations in PD mice, provide a critical foundation for future investigations into the mechanistic links between TCE exposure and PD pathogenesis.
    Keywords:  LC–MS/MS; N‐acetyl‐S‐(1,2‐dichlorovinyl)‐L‐cysteine (NAcDCVC); Parkinson's disease (PD); S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC); S‐(1,2‐dichlorovinyl)‐glutathione (DCVG); trichloroethylene (TCE)
    DOI:  https://doi.org/10.1002/rcm.10117
  13. Biomed Chromatogr. 2025 Sep;39(9): e70181
      Polyadenosine diphosphate-ribose polymerase 1 (PARP-1) and 2 (PARP-2) are key DNA repair enzymes that promote single-strand break repair via the base excision pathway. Niraparib, a PARP inhibitor, has shown clinical efficacy with the reduction of disease progression or death and progression-free survival benefit across multiple clinical trials, leading to the Food and Drug Administration (FDA) approval for the treatment of advanced and recurrent ovarian cancers. This study presents a robust and simple 5-min assay designed for the quantitation of the single agent niraparib in human plasma utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). A 50 μL volume of plasma was subjected to protein precipitation, followed by chromatographic separation using a Phenomenex Kinetex C18 column (2.6 μm, 50 × 2.1 mm) and a gradient mobile phase system consisting of 0.1% formic acid in both water and acetonitrile during a 5-min run time. Mass spectrometric detection was achieved using a SCIEX 6500 + tandem mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, our assay met the criteria outlined by the FDA guidance for bioanalytical method validation, demonstrating robust performance within the range from 5 to 5000 ng/mL. This assay will support future clinical studies by defining niraparib pharmacokinetics.
    Keywords:  LC‐MS/MS; human plasma; niraparib; pharmacokinetics; validation
    DOI:  https://doi.org/10.1002/bmc.70181
  14. Int J Mol Sci. 2025 Jul 21. pii: 6999. [Epub ahead of print]26(14):
      This study describes the development and validation of a fully automated workflow for serum sample preparation, enabling the quantitative determination of cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD, via liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Implemented on an automated platform, the workflow performs key steps such as solvent dispensing, mixing, centrifugation, filtration, and supernatant transfer, producing 96-well plates ready for analysis. Human serum samples were obtained from patients with epilepsy treated with CBD. All samples were processed using both manual and automated methods to evaluate method agreement. Quantification was performed by LC-MS/MS with CBD-d3 as the internal standard (IS). Method validation was conducted in accordance with European Medicine Agency (EMA) guidelines, confirming that the automated protocol meets the recommended acceptance criteria for both intraday and interday precision and accuracy. Calibration curves demonstrated excellent linearity across the concentration ranges. Comparative analysis using Passing-Bablok regression and Bland-Altman plots demonstrated strong agreement between the methods. These findings support the clinical applicability of the automated method for the therapeutic drug monitoring (TDM) of CBD and 7-hydroxy-CBD, and its robust performance and scalability provide a solid foundation for the development of an expanded analytical panel covering a broader range of antiseizure medications (ASMs), enabling more standardized TDM protocols in clinical practice.
    Keywords:  7-hydroxy-CBD; CBD; LC-MS/MS; TDM; automation; epilepsy
    DOI:  https://doi.org/10.3390/ijms26146999
  15. Anal Bioanal Chem. 2025 Jul 26.
      The achievement of a comprehensive profiling of plant metabolites has long represented a challenge, not only due to their wide-ranging abundances but also as a result of their considerable chemical diversity. Recent advances in highly sensitive liquid chromatographic (LC) techniques, particularly when coupled with high-resolution mass spectrometry (HRMS), have established metabolomics as a key approach for the analysis of thousands of non-volatile metabolites in crude natural extracts. Nevertheless, the different polarities of primary and secondary metabolites often limit the efficacy of conventional reversed-phase liquid chromatography (RPLC) in providing exhaustive compound coverage. To address this limitation, orthogonal separation techniques such as hydrophilic interaction liquid chromatography (HILIC) should be employed as a complement to RPLC. In this work, four columns with identical geometrical specifications but with different stationary phase chemistry (one reversed-phase C18 and three different HILIC adsorbents) were employed for the untargeted analysis of bioactive compounds contained in Hypericum perforatum. The columns were evaluated not only in terms of chromatographic performance but also based on their ability to resolve challenging isobaric compound pairs of isobaric compounds. Finally, the integration of RPLC and HILIC data enabled a more comprehensive characterization of the metabolites associated with the plant.
    Keywords:   Hypericum perforatum ; High-resolution mass spectrometry; Hydrophilic interaction liquid chromatography; Reversed-phase liquid chromatography; Untargeted analysis
    DOI:  https://doi.org/10.1007/s00216-025-06030-8
  16. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jul 23. pii: S1570-0232(25)00290-9. [Epub ahead of print]1264 124736
      Silicosis is characterized by the formation of fibrotic lesions due to altered collagen synthesis, in which amino acids play a crucial role. However, targeted metabolomics studies of serum amino acids in silicosis patients remain limited. Herein, we developed a specific serum amino acid metabolomics assay for identifying differential amino acids as biomarkers, providing a sensitive and reliable basis for diagnosing silicosis. A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous quantification of 21 amino acids in serum was established by optimizing chromatographic and sample preparation conditions. The method validation results showed good linearity (r > 0.99), recovery (78.3 %-128.4 %) and precision (1.27 %-13.01 %). The developed method was utilized to detect the levels of 21 amino acids in the serum of the silicosis patient group (SP; n = 104) and the healthy control group (HC; n = 118). The concentration of four amino acids (glutamate, arginine, aspartic acid and ornithine) was significantly different between the SP and the HC. Single diagnostic biomarkers exhibited area under the curve (AUC) values ranging from 0.712 to 0.892, while the glutamate-arginine combination achieved superior performance (AUC = 0.915, 86.1 % sensitivity, 90.0 % specificity). The aforementioned results indicate that four biomarkers were screened for the diagnosis of silicosis, with glutamate emerging as the most accurate single biomarker for predicting silicosis. The diagnostic performance was further enhanced by combining glutamate with arginine, which improved both sensitivity and specificity.
    Keywords:  Amino acids; Biomarker; Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS); Serum; Silicosis; Targeted metabolomics
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124736
  17. Anal Chem. 2025 Jul 29.
      High-resolution mass spectrometry imaging (MSI) plays a vital role in lipidomics, yet challenges persist in analyzing lipids at the single-cell level due to limitations in spatial resolution and lipid coverage. While existing strategies based on a single matrix application step for dual-polarity provide high lipid coverage from the same sample and enable easy sample preparation, matrix depletion limits their spatial resolution to 10 μm, preventing their application to single-cell imaging. Here, we present a single-cell/subcellular resolution strategy for dual-polarity matrix-assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) that eliminates the need for matrix reapplication. This approach achieves 5 μm spatial resolution while maintaining lipid coverage comparable to multistep single-cell imaging methods. This is enabled by a fine-tuned matrix deposition technique that fully utilizes the high sensitivity of N-(1-naphthyl)-ethylenediamine dihydrochloride (NEDC) in dual polarities and optimized acquisition conditions, allowing single-deposition workflows without the need for washing, repreparation, or image recalibration. This single-cell resolution MALDI-MSI strategy successfully imaged a broader range of lipid species with distinctive spatial detail in mouse kidney tissue and lung carcinoma cells (A549). Using spatial probabilistic latent semantic analysis (PLSA), we identified three distinct lipid distribution patterns within a single-cell population in both polarities, and histogram analysis revealed substantial cell-to-cell lipidomic heterogeneity. This strategy overcomes limitations of traditional dual-polarity MSI and provides a powerful tool for advancing cellular lipidomics, elucidating disease mechanisms, and investigating environmental toxicology.
    DOI:  https://doi.org/10.1021/acs.analchem.5c03289
  18. Proteomics. 2025 Jul 30. e70020
      Top-down mass spectrometry (TDMS) is the method of choice for analyzing intact proteoforms, as well as their posttranslational modifications and sequence variations. In top-down tandem mass spectrometry (TD-MS/MS) experiments, multiple proteoforms are often co-fragmented, resulting in multiplexed TD-MS/MS spectra. Due to their increased complexity, compared to spectra from single proteoforms, multiplexed TD-MS/MS spectra present significant challenges for proteoform identification and quantification. Here we present TopMPI, a new computational tool specifically designed for the identification of multiplexed TD-MS/MS spectra. Experimental results demonstrate that TopMPI substantially increases the sensitivity and accuracy of proteoform identification in multiplexed TD-MS/MS spectral analysis compared to existing tools. SUMMARY: Top-down mass spectrometry (TDMS) is a powerful technique for analyzing intact proteoforms; however, identifying multiple co-fragmented proteoforms from multiplexed tandem mass spectrometry (MS/MS) spectra remains a significant challenge. In this paper, we introduce TopMPI, a new computational tool specifically designed to identify multiplexed TD-MS/MS spectra using a two-round database search strategy. Compared to existing tools, TopMPI significantly improves the sensitivity and accuracy of proteoform identification from multiplexed MS/MS spectra. The development of TopMPI enhances the identification of low abundance proteoforms in complex biological samples and increases the potential of TDMS for discovering proteoform biomarkers in disease studies.
    Keywords:  database searching; multiplexing; proteoform identification; top‐down proteomics
    DOI:  https://doi.org/10.1002/pmic.70020
  19. J Pharm Sci. 2025 Jul 25. pii: S0022-3549(25)00379-X. [Epub ahead of print] 103927
      Currently, the world health organization (WHO) recommends a fixed-dose combination therapy called TLD, composed of tenofovir, lamivudine, and dolutegravir (TLD) for human immunodeficiency virus (HIV) treatment. About 23 of 39 million people living with HIV/AIDS (PLWHA) are on a daily TLD pill. In this study, we successfully developed a simple, efficient, and sensitive method to extract and enable the simultaneous quantification of tenofovir, lamivudine, and dolutegravir in rats, non-human primates (NHP), and human plasma samples. The 3 HIV drugs in small volume, 100 µL plasma were extracted after protein precipitation, and all the analytes were quantified using a novel liquid chromatography-tandem mass spectrometry. The assay provided analysis of all three analytes in rat, NHP, and human plasma. This method was used to evaluate time-plasma drug concentrations in two NHPs (Macaca nemestrina). The reported bioanalytical method demonstrated high sensitivity, reproducibility, and robustness for quantifying the 3 HIV drugs with efficiency. Lower limit of quantification (LLOQ) for tenofovir (TFV), lamivudine (3TC) and dolutegravir (DTG) were between 0.3 - 2.6 ng/mL equivalent to 30 - 260 pg in 100 µL of rats, NHPs, or human plasma samples. This assay may be adapted for evaluating drug concentrations across multiple species to support preclinical, clinical, and translational studies for developing long-acting products.
    Keywords:  Antiinfective(s); Drug-combination particle(s); HIV/AIDS; Injectable(s); Liquid Chromatography-Mass Spectrometry (LC-MS); Longacting; Mass spectrometry; Preclinical pharmacokinetics; bioanalysis; chromatography
    DOI:  https://doi.org/10.1016/j.xphs.2025.103927
  20. Talanta. 2025 Jul 21. pii: S0039-9140(25)01096-3. [Epub ahead of print]297(Pt A): 128606
      Alpha-fetoprotein (AFP) is a critical biomarker widely used for the screening and diagnosis of hepatocellular carcinoma and germ cell tumors. Although immunoassays are commonly employed for AFP detection, significant discrepancies in measurement results across different laboratories persist due to poor harmonization among methods. To improve the accuracy and harmonization of AFP quantification, this study developed a novel magnetic solid-phase extraction coupled with isotope dilution liquid chromatography-tandem mass spectrometry (MSPE-ID-LC-MS/MS) method for the precise measurement of AFP in serum. The method utilizes high-affinity AFP antibodies cocktails conjugated with magnetic nanoparticles to enhance the recovery of low-abundance AFP, combined with optimized elution conditions to ensure analytical reliability. The protocol involves one-step denaturation and alkylation, followed by tryptic digestion. This enables accurate quantification with SI-traceability using three signature peptides. Method validation demonstrated that intra- and inter-day precisions were <10 %, recovery rates of 99.8-101.4 %, and the limit of quantification (LOQ) of 1.5 ng/mL. This work establishes a metrologically robust reference procedure for AFP measurement and paves the way for standardization of clinical protein assays.
    Keywords:  Alpha-fetoprotein; Isotope dilution; LC-MS/MS; Quantification; Reference method; Standardization
    DOI:  https://doi.org/10.1016/j.talanta.2025.128606
  21. Talanta. 2025 Jul 24. pii: S0039-9140(25)01118-X. [Epub ahead of print]297(Pt A): 128627
      Circadian rhythm (CR) in humans regulates the physical and mental changes that take place following rhythmic shifts of light and darkness. Dysregulation of CR is related to sleep disorders and other health problems including immune suppression, cancer, neurological and cardiovascular diseases. The two main hormones that maintain a balance to control the sleep-wake cycles are melatonin (MEL) and cortisol (COR). Here we describe a novel environmentally friendly low-density solvent-dispersive liquid-liquid microextraction-ultra-performance liquid chromatography-tandem mass spectrometry (LDS-DLLME-UPLC-MS/MS) method for simultaneous quantification of MEL, COR and their metabolites: 2-hydroxymelatonin (2-HMEL), 6-hydroxymelatonin (6-HMEL), cyclic-3-hydroxymelatonin (3-cHMEL), N-γ-acetyl-5-methoxykynurenamine (AMK), N-acetyl-N-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), 6-sulfatoxy melatonin (SaMT), 6-hydroxycortisol (6-HCOR), cortisol sulfate (CORS), cortisone (CON), β-cortol (CO) and β-cortolone (COL) in urine. Chemometrics approaches were applied for method optimization. The method was validated with the recovery of target analytes at 100 %, precision <16 % RSD and the absence of matrix effects. The method limits of quantification (LOQs) were from 0.013 ng mL-1 for MEL to 0.79 ng mL-1 for COL. This novel method was applied for the analysis of real urine samples. All targeted biomarkers were detected in urine samples, except AMK. MEL, 2-HMEL, 3-cHMEL, 6-HMEL, AFMK, NAS, COL and CO were found predominantly as glucuronidated conjugates, whereas SaMT, CORS and 6-HCOR were detected in free form in urine. We observed remarkable variation in profiles of MEL and COR metabolites in first morning void, daytime and nighttime urine samples, suggesting that this method can be applied for monitoring circadian rhythm in adults and children. The most sensitive urinary biomarkers of CR are 2-HMEL, 6-HMEL, SaMT, COR, 6-HCOR and CON.
    Keywords:  Circadian; Cortisol; DLLME; Green chemistry; LC-MS/MS; Melatonin; Urine
    DOI:  https://doi.org/10.1016/j.talanta.2025.128627
  22. Bioanalysis. 2025 Jul 29. 1-14
       AIMS: This study aims to address the clinical need for managing secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD) patients on dialysis and hypercalcemia in individuals with parathyroid carcinoma or primary hyperparathyroidism (PHPT) ineligible for parathyroidectomy. Cinacalcet, a key calcimimetic agent, targets the calcium-sensing receptor (CaSR) in the parathyroid gland to lower elevated parathyroid hormone (PTH) levels. The study focuses on developing a robust high-performance liquid chromatography tandem mass spectrometry method for cinacalcet quantification to support therapeutic monitoring and pharmacokinetics.
    METHODS: A bioanalytical method using cinacalcet-D3 (CCT-D3) as an internal standard and tandem mass spectrometry in positive ion mode for accurate quantification employing Triple Quad Mass spectrometer in normal mode and a Zorbax Eclipse XDB-C18 column.
    RESULTS: The method was comprehensively validated, demonstrating reliability through high precision, accuracy, linearity within the range of 0.300-150.00 ng/mL, and stability, all in compliance with established bioanalytical guidelines. Furthermore, its environmental sustainability was assessed using modern green chemistry evaluation metrics.
    CONCLUSION: This study presents a robust, selective LC-MS/MS assay for quantifying cinacalcet in human plasma. The assay demonstrates excellent linearity, precision, and recovery, making it suitable for pharmacokinetic studies, therapeutic drug monitoring, and bioequivalence or bioavailability assessments.
    Keywords:  Bioequivalence; ICH M10 guidelines; cinacalcet; high-performance liquid chromatography with tandem mass spectrometry; liquid-liquid extraction
    DOI:  https://doi.org/10.1080/17576180.2025.2535945
  23. MethodsX. 2025 Dec;15 103495
      Ergosterol is widely used as proxy for the estimation of leaf litter associated fungal biomass. According to a common textbook method, ergosterol can be extracted from plant tissue with methanolic potassium hydroxide followed by purification with solid-phase extraction (SPE) and detection via high-performance liquid chromatography-ultraviolet (HPLC-UV). As an alternative, we developed a method using liquid-liquid extraction (LLE) of the methanolic extract with cyclohexane instead of time consuming and error prone SPE. For ergosterol detection, HPLC-UV was replaced by the more sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated for alder leaves at 10 µg/g dw (LOQ) and 500 µg/g dw with mean recoveries between 95.1 and 100.2 % (relative standard deviations < 10 %) and has been successfully applied for the measurement of fungicide effects on leaf litter associated aquatic fungi.•Selective and flexible analytical method for ergosterol quantification in alder leaves using LLE and LC-MS/MS instead of SPE and HPLC-UV•Optional use of 7-dehydrocholesterol as internal standard to allow for compensation of volumetric variations during sample preparation and signal drift in LC-MS/MS•Method validation for three mass transitions according to guidance document SANTE/2020/12830 including matrix effect evaluation and stability investigations.
    Keywords:  Cryo-milling; Fungal biomarker; Fungal biomass; Heat-reflux extraction; Leaf litter decomposition; Miniaturized sample preparation; Semi-closed digestion unit
    DOI:  https://doi.org/10.1016/j.mex.2025.103495
  24. Pharmaceuticals (Basel). 2025 Jul 02. pii: 998. [Epub ahead of print]18(7):
      Background/Objectives: We developed and validated a robust and simple LC-MS/MS method for the simultaneous quantification of amoxicillin and clavulanate in human plasma relative to previously reported methods. Methods: Amoxicillin; clavulanate; and an internal standard, 4-hydroxytolbutamide, in human K2-EDTA plasma, were deproteinized with acetonitrile and then subjected to back-extraction using distilled water-dichloromethane. Separation was performed on a Poroshell 120 EC-C18 column with a mobile-phase gradient comprising 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.5 mL/min within 6.5 min. The negative electrospray ionization modes were utilized to monitor the transitions of m/z 363.9→223.1 (amoxicillin), m/z 198.0→135.8 (clavulanate), and m/z 285.0→185.8 (4-hydroxytolbutamide). Results/Conclusions: Calibration curves exhibited linear ranges of 10-15,000 ng/mL for amoxicillin (r ≥ 0.9945) and 20-10,000 ng/mL for clavulanate (r ≥ 0.9959). Intra- and inter-day's coefficients of variation, indicating the precision of the assay, were ≤7.08% for amoxicillin and ≤10.7% for clavulanate, and relative errors in accuracy ranged from -1.26% to 10.9% for amoxicillin and from -4.41% to 8.73% for clavulanate. All other validation results met regulatory criteria. Partial validation in lithium-heparin, sodium-heparin, and K3-EDTA plasma confirmed applicability in multicenter or large-scale studies. This assay demonstrated itself to be environmentally friendly, as assessed by the Analytical GREEnness (AGREE) tool, and was successfully applied to a clinical pharmacokinetic study of an Augmentin® IR tablet (250/125 mg). The inter-individual variabilities in clavulanate exposures (AUCt and Cmax) were significantly greater than in amoxicillin, and they may inform the clinical design of future drug-drug interaction.
    Keywords:  LC–MS/MS; amoxicillin; clavulanate; human K2-EDTA plasma; pharmacokinetics; validation
    DOI:  https://doi.org/10.3390/ph18070998
  25. J Chromatogr A. 2025 Jul 19. pii: S0021-9673(25)00580-1. [Epub ahead of print]1759 466235
      Human biomonitoring of persistent organic pollutants (POPs) remains essential for tracking long-term exposure, evaluating health risks, and assessing the effectiveness of regulatory bans. For this purpose, an experimental and analytical methodology has been optimized allowing the determination of 47 POPs, encompassing chlorinated cyclodienes, chlorobenzenes, cyclohexanes, polychlorodiphenyl derivatives, several congeners of polychlorobiphenyls (PCBs) and polybromodiphenyl ethers (PBDEs, including the deca-BDE), as well as three non-persistent chemicals (namely quintozene, tecnazene and vinclozolin). The method uses a single liquid-liquid extraction procedure with 500 µL of serum/plasma. Instrumental analysis involves gas chromatography (GC) with electron capture detection (ECD) for the analysis of organochlorines, and a subsequent injection into a GC coupled to mass spectrometry in negative chemical ionization mode (MS-NCI) for the determination of PBDEs. The analytical procedure has been successfully validated, demonstrating high sensitivity (limits of detection: 0.0016-0.012 ng/mL; limits of quantification: 0.0029-0.021 ng/mL), acceptable linearity (R² > 0.9962), good precision (coefficients of variation <25%, except for BDE-209), satisfactory accuracy (recoveries within 70-130% for 41 out of 47 compounds) and minimal matrix effects. Proficiency Testing Materials from the AMAP Ring Test for POPs in human serum confirmed the method's reliability with results within the accepted reference range. This method represents an efficient and practical approach for human biomonitoring studies, with a simplified workflow suitable for routine application.
    Keywords:  Deca-BDE; Gas chromatography and mass spectrometry; Human Biomonitoring; Human serum/plasma; Persistent organic pollutants; Vinclozolin
    DOI:  https://doi.org/10.1016/j.chroma.2025.466235
  26. Pharmaceutics. 2025 Jul 16. pii: 919. [Epub ahead of print]17(7):
      Background/Objectives: Selinexor is a selective nuclear-export inhibitor approved for hematologic malignancies, characterized by extensive plasma protein binding (>95%). However, a validated analytical method to accurately measure the clinically relevant unbound fraction of selinexor in human plasma has not yet been established. This study aimed to develop a fully validated bioanalytical assay for simultaneous quantification of total and unbound selinexor concentrations in human plasma. Methods: We established and fully validated an analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) capable of quantifying total and unbound selinexor concentrations in human plasma. Unbound selinexor was separated using ultrafiltration, and selinexor was efficiently extracted from 50 μL of plasma by liquid-liquid extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase (0.1% formic acid:methanol, 12:88 v/v) with a relatively short runtime of 2.5 min. Results: Calibration curves showed excellent linearity over a range of 5-2000 ng/mL for total selinexor (r2 ≥ 0.998) and 0.05-20 ng/mL for unbound selinexor (r2 ≥ 0.995). The precision (%CV ≤ 10.35%) and accuracy (92.5-104.3%) for both analytes met the regulatory criteria. This method successfully quantified selinexor in plasma samples from renally impaired patients with multiple myeloma, demonstrating potential inter-individual differences in unbound drug concentrations. Conclusions: This validated bioanalytical assay enables precise clinical pharmacokinetic assessments in a short runtime using a small plasma volume and, thus, assists in individualized dosing of selinexor, particularly for renally impaired patients with altered protein binding.
    Keywords:  LC-MS/MS; bioanalytical method; human plasma; multiple myeloma; protein binding; selinexor; validation
    DOI:  https://doi.org/10.3390/pharmaceutics17070919
  27. Metabolomics. 2025 Jul 27. 21(4): 103
       INTRODUCTION: Untargeted metabolomics is a popular method by which researchers measure a large portion of the metabolites present in a biological system at once. This approach usually results in complex data sets containing tens to hundreds of thousands of observations which require sophisticated data analysis workflows. To help with the functional interpretation of the data, researchers often rely on enrichment analysis. However, little advice is available on what method to use, and, to the best of our knowledge, there is no comparison of popular approaches available for in vitro data with a focus on toxicological and pharmacological testing.
    OBJECTIVES: In this study, we compared three popular enrichment analysis approaches-Metabolite Set Enrichment Analysis (MSEA), Mummichog and Over Representation Analysis (ORA)-with data obtained by treating Hep-G2 cells with 11 compounds with five different mechanisms of action. We compared the results and assessed the consistency of the individual methods as well as their correctness.
    METHODS: Hep-G2 cells were treated with subtoxic concentrations of 11 test compounds. After preparation, samples were measured on an Elute UHPLC coupled to a timsTOF Pro (both Bruker). Spectra were processed in MetaboScape (Bruker) and annotated using spectral library search. Datasets were further processed using R and enrichment analysis was performed in MetaboAnalyst.
    RESULTS: Overall, we observed a low to moderate similarity between different enrichment methods with the highest similarity between MSEA and Mummichog. Further, Mummichog outperformed both MSEA and ORA in terms of consistency and correctness.
    CONCLUSION: In our comparison, Mummichog showed the best performance for in vitro untargeted metabolomics data.
    DOI:  https://doi.org/10.1007/s11306-025-02309-0
  28. Angew Chem Int Ed Engl. 2025 Jul 29. e202507483
      Derivatization-enhanced multidimensional metabolomics combined with ion mobility mass spectrometry will greatly improve the accuracy and coverage of metabolic analysis. However, accurate prediction of the large-scale collision cross section (CCS) of derivatized metabolites without relying on standards and the establishment of multidimensional analytical methods faces great challenges. Here, we propose quantum chemistry calculation-assisted machine learning strategies applicable to the accurate prediction of the CCS of derivatized sterols, develop C═C bond-targeted N-Me derivatization methods for unsaturated sterols, and create a large-scale, 4D information database of derivatized sterol lipids (n = 4891) by combining retention time and fragment ion prediction. Furthermore, a high-coverage unsaturated sterolomics at the isomer level was established on this basis, which quantitatively revealed the tissue-specific distribution patterns of over 100 sterol lipids. This study provides a key foundation for derivatization-enhanced metabolomics and provides important techniques and information for metabolic and functional studies of sterols.
    Keywords:  Lipids; Machine learning; Mass spectrometry; Quantum chemistry; Sterolomics
    DOI:  https://doi.org/10.1002/anie.202507483
  29. Bioanalysis. 2025 Jul 30. 1-9
       BACKGROUND: Current HPLC-based methods for doxofylline analysis lack speed and precision. A rapid, specific, and sensitive UPLC-MS/MS method was developed for the determination of doxofylline in this study.
    RESEARCH DESIGN AND METHODS: This method was fully validated and doxophylline-d4 was used as an internal standard. A Kinetex-C18 column (EVO 100Å, 50 × 2.1 mm, 5 μm) was used for the separation procedure, with mobile phases consisting of 0.3% formic acid (A) and 90% acetonitrile solution with 0.3% formic acid (B). The total runtime of the gradient elution procedure was 2.6 minutes. The mass spectrometry analysis was carried out employing a multiple reaction monitoring model and using the transitions of m/z 267.000→181.000 for doxofylline and m/z 271.200→181.100 for the internal standard.
    RESULTS: The linear range of detection for doxophylline was between 20.0 to 16,000 ng/mL. The intra-batch accuracy deviations of each concentration level ranged from -8.0% to 2.5%, while the intra-batch precisions ranged from 1.3% to 9.0%. And the inter-batch accuracy deviations were -5.8% ~0.8%, while the inter-batch precisions were 2.2% ~7.0%.
    CONCLUSIONS: This method was applied to pharmacokinetic clinical trials of single oral administration of doxophylline tablets successfully.
    CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov identifier is CTR20240006 and CTR20233665.
    Keywords:  Doxofylline; UPLC-MS; doxophylline-d4; pharmacokinetic; validation
    DOI:  https://doi.org/10.1080/17576180.2025.2535950
  30. Talanta. 2025 Jul 21. pii: S0039-9140(25)01101-4. [Epub ahead of print]297(Pt A): 128611
      At present, mass spectrometry (MS) and mass spectrometry imaging (MSI) have developed into versatile analytical techniques, applicable for the analysis of both small molecules and macromolecular compounds and complexes. Desorption electrospray ionization (DESI), as an ambient MS (AMS) technique, enables rapid analysis and imaging under open conditions with minimal or no sample preparation required. In this review, the principles and mechanisms of DESI were described, especially for the factors affecting the analysis including suppression effects, device condition parameter settings, and ionization and transmission efficiency. Moreover, the relevant improvements of DESI technique were introduced in detail. Emphasis has been made to discuss the recent research on the application of DESI MS and MSI in the fields of metabolomics, lipidomics, and proteomics. In these applications, DESI MS and MSI exhibit exceptional capabilities for qualitative analysis.
    Keywords:  Desorption electrospray ionization; Lipidomics; Mass spectrometry; Mass spectrometry imaging; Metabolomics; Proteomics
    DOI:  https://doi.org/10.1016/j.talanta.2025.128611
  31. Commun Chem. 2025 Aug 01. 8(1): 224
      Due to the potential health risks related to chemical exposure, rapidly assessing xenobiotic molecules in the environment and those already in the body is imperative. Targeted analytical methods coupling either gas or liquid chromatography with mass spectrometry (GC-MS or LC-MS) are commonly utilized in current exposure assessments. While these methods are accepted as the gold standard for exposure analyses, they often require multiple sample preparation steps and analysis times > 30 min. These limitations have resulted in an evolving interest in using ion mobility spectrometry and MS (IMS-MS), either with or without chromatography, to improve throughput and annotation confidence. To increase IMS-MS information availability for exposure studies, here we utilized drift tube IMS-MS to evaluate 4685 xenobiotic chemical standards from the Environmental Protection Agency Toxicity Forecaster (ToxCast) programme, including pesticides, industrial chemicals, pharmaceuticals, consumer products, and per- and polyfluoroalkyl substances. Collision cross section (CCS) and m/z values were detected for 2144 unique chemicals with high confidence and reproducibility (≤1% error intra-laboratory and ≤2% inter-laboratory), resulting in values for 4004 [M + H]+, [M+Na]+, [M-H]- and [M]•+ ion types. This multidimensional database therefore supports suspect screening for a wider range of environmental contaminants, faster exposure response times, and assessments of xenobiotic-disease connections.
    DOI:  https://doi.org/10.1038/s42004-025-01619-7
  32. Int J Microbiol. 2025 ;2025 4388417
      Prokaryotic organisms rely on a limited array of metabolites for survival, which varies according to their natural environment. For example, soil-borne bacteria produce diverse metabolites, such as antibiotics, to thrive in their competitive surroundings, inhibiting the growth of nearby competing bacteria. The structural diversity of these compounds offers great analytical challenges, since there is no universal acquisition setting that can be applied to achieve their comprehensive coverage. Therefore, the use of a single experimental setup inevitably hinders the comprehensive metabolite coverage, which would affect the outputs. To address this, we propose employing a design of experiment (DoE) approach through the central composite design (CCD) to enhance the metabolite detection and broaden the coverage of the data-dependent acquisition (DDA) mode of the UHPLC-qTOF-MS technique. Our study reveals that altering collision energy significantly enhances metabolite coverage compared to adjusting the DDA threshold of detection. Furthermore, the ability of global natural product social (GNPS)-based molecular network models to annotate metabolites is greatly influenced by data acquisition settings, particularly affecting MS2 data. Interestingly, molecular networks constructed from averaged spectral data obtained through randomly selected DDA settings outperform those generated using customized settings through DoE modeling. This study demonstrates that in untargeted LC-MS metabolomics, both collision energy and intensity threshold independently enhance metabolite coverage in untargeted metabolomics. However, their combined use results in even greater coverage. Consequently, we recommend adopting group-based optimization over single-point optimization for more comprehensive metabolite coverage and in-depth exploration. However, caution should be taken in order to balance between robust data and redundancy.
    Keywords:  LC-MS (liquid chromatography-mass spectrometry); bacterial metabolites; data-dependent acquisition (DDA); metabolite profiling; metabolomics; molecular networking
    DOI:  https://doi.org/10.1155/ijm/4388417
  33. Forensic Toxicol. 2025 Aug 01.
       PURPOSE: Over-the-counter medicines are commonly used for recreational and suicidal overdoses, a global problem. Some of these are easily obtained via the Internet. In cases of intoxication, drug quantification is necessary to estimate the cause of death. Stable isotope compounds are recommended as internal standards (IS) for analyzing drugs; however, it is difficult for individual laboratories to obtain isotopes for all analytes due to cost and availability. Therefore, alternative IS selection is important for practicality. Here, we quantified diphenhydramine and dextromethorphan concentrations in plasma from several collection sites in a fatal intoxication case, and assessed various IS performance based on structural similarities and retention time.
    METHODS: A mid-teenager died from intoxication of personally imported dextromethorphan and Over-the-counter diphenhydramine. To quantify these drugs, we selected morphine-d3, dihydrocodeine, diphenhydramine-d3, mianserin-d3, and diazepam-d5 as alternative IS and evaluated. After selecting the most suitable IS, we quantified dextromethorphan and diphenhydramine concentrations in twelve plasma samples from the victim by liquid chromatography-tandem mass spectrometry.
    RESULTS: Recovery rates were 80.7-105.5%, except for morphine-d3 (47.8%) and dihydrocodeine (64.8%). Matrix effects were 75.7-103.2%. The intra-day accuracies and precisions were 86.4-119.5% and 0.27-12.2%, respectively. The inter-day accuracies were 81.2-119.8%, and the precisions were 0.80-9.44%. The validation study showed that diphenhydramine-d3 was the most suitable IS. Finally, plasma concentrations of dextromethorphan and diphenhydramine were 3.74-10.3 µg/mL and 15.6-52.9 µg/mL, respectively.
    CONCLUSIONS: The concentrations of both drugs in plasma samples were estimated to cause death. When using an alternative IS, a validation study is needed to select the optimal IS.
    Keywords:  Dextromethorphan; Diphenhydramine; Internal standards; LC/MS/MS; Liquid chromatography-tandem mass spectrometry; Overdose
    DOI:  https://doi.org/10.1007/s11419-025-00736-1
  34. Anal Chem. 2025 Jul 30.
      Lipid regulation and remodeling are pivotal in follicle development and oocyte maturation. Performing lipidome analysis on single oocytes is essential for optimizing assisted reproductive techniques and improving pregnancy outcomes. Conventional organic solvent extraction faces challenges in single-cell analysis, including excessive sample dilution and time-consuming processing. In this study, we developed an on-valve microflow supercritical fluid extraction and chromatography-mass spectrometry (μSFE-SFC-MS) method. Lipids in intact single oocytes are extracted online by supercritical carbon dioxide fluid, separated by a C18 capillary column, ionized, and annotated. Notably, this method significantly reduces sample pretreatment and chromatographic separation time to just 15 min per sample, compared to about 2 h of the conventional method. We analyzed of 276 lipid species from a single oocyte. Lipidomic differences in oocyte maturation stages clarify distinct metabolic remodeling of phospholipids into sphingomyelins and glycerides. The differential analysis suggests that several lipid species can be used as criteria for determining oocyte maturation at the single-cell level. The present work offers a fast and high-coverage lipidomic analysis for single oocytes, also providing a workflow for other single-cell and trace samples.
    DOI:  https://doi.org/10.1021/acs.analchem.5c03365
  35. J Am Soc Mass Spectrom. 2025 Jul 28.
      The ability to differentiate disaccharide isomers within plant tissues, in a spatially resolved fashion, is required to render a more precise understanding of their biological function within these systems. In this study, we report drift tube ion mobility spectrometry (DTIMS) and trapped ion mobility spectrometry (TIMS) profiling of two important plant disaccharides, sucrose and trehalose, along with their 6-phosphate derivatives. We then map these disaccharides in poplar root and soybean root nodules using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) with premass analysis TIMS. This approach bridged an analytical gap for the straightforward visualization and discrimination of disaccharides within plant tissues at a spatial resolutions approaching a single plant cell size. We found direct infusion DTIMS and TIMS MS worked congruently to obtain the collision cross section (CCS) values of disaccharides, where we measured ΔCCS < 1.5% between DTCCSN2 and TIMSCCSN2 across all ions compared. This approach provided a baseline comparison for subsequent MSI measurements. We observed the disaccharide ion at m/z 381.08 ([M + K]+) displayed a single CCS value in poplar roots, whereas two different CCS values were measurable in soybean root nodules, which exemplified coexisting disaccharide isomers in this plant root tissue. We found the high-resolution ion mobility fingerprinting and ion imaging for the disaccharide isomer 6-phosphate derivatives in plant tissues were more challenging compared to those for the nonphosphorylated disaccharide species. This may be in part due to their lower abundance and MALDI-induced in-source fragmentation. Empowered by the integration of ion mobility spectrometry and MSI, this study provides a new avenue for easier characterization and direct visualization of disaccharide isomers within plant tissue.
    DOI:  https://doi.org/10.1021/jasms.5c00101
  36. Ann Clin Lab Sci. 2025 May;55(3): 449-452
       OBJECTIVE: Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) is the gold standard for the analysis of steroids including 11-deoxycortisol, androstenedione, testosterone, 17-hydroxyprogesterone, and dehydroepiandrosterone (DHEA) in serum or plasma. In our hospital we observed an occasional problem in analysis of testosterone in some patients when blood was collected in Beckton-Dickinson serum separator gel tubes (BD gel tubes). Therefore, we investigated this issue.
    METHODS: Blood was collected from 10 volunteers (five males and five females) simultaneously in plain red top tubes (no gel), Greiner serum separator gel tubes (Greiner gel tube), and BD (Beckton-Dickinson) gel tubes followed by extraction of steroids from serum, reconstitution with mobile phase, and subsequent analysis of various steroids using LC-MS/MS in a single run.
    RESULTS: No interfering peak was observed when blood was collected in plain red top tube for all five steroids. However, we observed clinically significant interference with low testosterone values when blood specimens were collected in BD gel tubes. For DHEA, we observed interference when blood was collected in BD gel tubes and Griner tubes. Other steroids were not affected.
    CONCLUSIONS: We concluded that BD serum separator gel tube may not be suitable for analysis of testosterone in women and DHEA for both men and women.
    Keywords:  17-hydroxyprogesterone; Beckton-Dickinson serum separator gel; Dehydroepiandrosterone; Greiner serum separator gel; LC-MS/MS; testosterone
  37. Metabolomics. 2025 Jul 27. 21(4): 101
       INTRODUCTION: Untargeted metabolomics is a powerful tool for detecting perturbations in biological systems, offering significant potential for screening for rare inherited metabolic disorders (IMDs). However, the rarity and vast diversity of these diseases, results in limited availability of samples and incomplete metabolic pathway knowledge for each condition. Current diagnostic procedures rely heavily on manual interpretation, which is time-consuming, and data driven approaches are insufficient for small sample sizes.
    OBJECTIVES: To develop a diagnostic algorithm for IMDs addressing the challenges posed by small sample sizes and continuously evolving datasets.
    METHODS: 77 IMD patients (35 different IMDs) and 136 control samples were collected from Copenhagen University Hospital, Rigshospitalet. The metabolome was analyzed using liquid chromatography-mass spectrometry. An algorithm partially based on sparse hierarchical clustering was designed to generate IMD-specific metabolic signatures from metabolomics data, enabling comparison with undiagnosed patient samples to provide diagnostic predictions. An iterative improvement strategy was employed, where new data are continuously integrated to refine the IMD-specific signatures. The algorithm's performance was evaluated through both the current study and a case study using literature-derived data.
    RESULTS: The algorithm demonstrated iterative improvement with each training round, correctly identifying the diagnosis within top 3 potential IMDs in 60% of samples (top 1 in 42%). The case study applied the method to literature-based data comprising 95 IMD samples (11 different IMDs) and 68 controls, yielding a correct diagnosis in 73.5% of cases.
    CONCLUSION: These results demonstrate that the algorithm provides a flexible, data-driven framework for continuous improvement in IMD diagnosis, even with limited number of samples.
    Keywords:  Diagnosis; IMD signature; Inherited metabolic disorders; Metabolomics; Unsupervised
    DOI:  https://doi.org/10.1007/s11306-025-02302-7
  38. Anal Chem. 2025 Jul 30.
      Tandem mass spectrometry (MS/MS) provides essential structural information and plays a central role in compound annotation in metabolomics. While different precursor ion types are expected to influence the generation of MS/MS spectra, systematic investigations into precursor ion type-dependent MS/MS variability have been limited. To address this gap, we analyzed over half a million MS/MS spectra of 24,686 unique compounds from the NIST 20 spectral library, covering a broad range of precursor ion types and collision energies (CEs). Using [M + H]+ and [M - H]- spectra as references, we found that alkali cation adducted species such as [M + Na]+ and [M + K]+ exhibited distinct fragmentation behavior and low spectral similarity, likely due to the distinct nature of the alkali charge carriers, which do not promote protonated fragmentation pathways but instead stabilize the precursor ion through coordination. In contrast, [M + NH4]+, [2M + H]+, [M + H - H2O]+, [M + Cl]-, [2M - H]-, and [M - H - H2O]- showed moderate to high similarity to their references, as they often undergo neutral losses that generate [M + H]+ or [M - H]-, or are themselves derived from these ions. Our study also observed that fragmentation is structure-driven at lower CE and energy-driven at higher CE. This pattern allows for a higher spectral similarity among different precursor ion types at high CE. However, [2M + H]+ or [2M - H]- showed reduced similarity at higher CE, likely because the same amount of energy is distributed across more bonds in these larger precursor ions, resulting in less energy per bond. Finally, we demonstrated that ignoring precursor ion types can compromise compound annotation, including spectral library searches, molecular networking, and machine learning model development. Overall, this study underscores the critical influence of precursor ion types on MS/MS spectra and highlights the need for precursor-ion-type-aware strategies in metabolite annotation, which has been largely overlooked in the metabolomics field.
    DOI:  https://doi.org/10.1021/acs.analchem.5c02792
  39. Anal Bioanal Chem. 2025 Jul 26.
      A novel method combining electroless ionisation mass spectrometry (ELI-MS) with solid-phase extraction (SPE) cartridges enables direct analysis of beta-agonist residues in bovine urine (SPE-ELI-MS). Using a syringe plunger and a battery-powered DIY syringe pump mounted on an XYZ-linear stage, rapid sample clean-up and precise positioning were achieved. Optimisation of spray solvent and nozzle placement improved performance, yielding high sensitivity, repeatability, linearity, and trueness with internal standard correction. This approach offers a simple, efficient solution for analysing organic residues in biological samples, with strong potential for future on-site applications.
    Keywords:  Ambient ionisation mass spectrometry; Beta-agonists; Bovine urine; ELI-MS; Electroless ionisation; Solid-phase extraction
    DOI:  https://doi.org/10.1007/s00216-025-06019-3
  40. Malar J. 2025 Jul 25. 24(1): 243
       BACKGROUND: Malaria still poses a significant burden on global health, with millions of cases reported annually and rising resistance to current treatments, emphasizing the need for new therapeutic strategies. Fosmidomycin, initially recognized for its antibacterial properties, has emerged as a promising candidate in the fight against malaria.
    METHODS: In this study, a sensitive and robust LC-MS/MS method for quantifying fosmidomycin in human and rat plasma was developed and validated. Plasma samples were prepared using a simple protein precipitation method with 10% trichloroacetic acid (TCA). The assay featured a rapid run time of 5 min, and validation was performed according to the European Medicines Agency's guidelines.
    RESULTS: The method validation confirmed its selectivity, linearity, accuracy, precision, and stability. Notably, the calibration range was established from 0.25 to 15 mg/L, demonstrating improvements over previous methodologies with lower limits of quantification of 0.5-1.0 mg/L. Using the developed LC-MS/MS method, plasma samples were analysed from a clinical trial conducted in Gabon, as well as from a pharmacokinetic study involving male Wistar rats, revealing viable pharmacokinetic profiles for fosmidomycin.
    CONCLUSIONS: These findings confirm the utility of the developed analytical method for supporting the clinical development of fosmidomycin as a potential therapy for malaria.
    Keywords:  Bioanalysis; Fosmidomycin; LC–MS/MS; Malaria; Pharmacokinetics
    DOI:  https://doi.org/10.1186/s12936-025-05489-1
  41. Gigascience. 2025 Jan 06. pii: giaf075. [Epub ahead of print]14
       BACKGROUND: The growing number of metabolomics studies, based on high-dimensional data measured by hyphenated mass spectrometry (MS) and/or nuclear magnetic resonance (NMR) spectroscopy, has sparked the creation of several public metabolomics data repositories. Each repository emphasizes different aspects regarding data selection and representation, but most offer only limited options for privacy-preserving data sharing.
    RESULTS: We present MetaboSERV, an open-source, browser-based metabolomics platform dedicated to the selection, integration, and sharing of quantitative metabolomics data and metadata with controlled data access. MetaboSERV aims to aid researchers in analyzing their results by facilitating means to browse, visualize, and compare data across available datasets. It provides different access control functionalities, creating an environment in which data can be shared safely in a privacy-preserving manner to support collaborative and interdisciplinary research. Furthermore, it is designed to be extensible and adaptable to existing data management infrastructures through the creation of self-managed MetaboSERV instances, for which we provide the source code and a set of configurable Docker images.
    CONCLUSIONS: The public MetaboSERV instance is available at https://metaboserv.ckdn.app, and the source code can be found at https://gitlab.gwdg.de/MedBioinf/metabolomics/metaboserv. The Research Resource Identifier (RRID) for MetaboSERV is SCR_025496.
    Keywords:  (privacy-preserving) data sharing; collaborative research; mass spectrometry; metabolomics; nuclear magnetic resonance spectroscopy
    DOI:  https://doi.org/10.1093/gigascience/giaf075
  42. Anal Chim Acta. 2025 Oct 08. pii: S0003-2670(25)00763-9. [Epub ahead of print]1370 344369
       BACKGROUND: Melatonin, a hormone synthesized by the pineal gland, exhibits immunomodulatory, anti-inflammatory, anti-tumor, antioxidant, and chronobiological properties. In breast milk, melatonin concentration has been related to influencing neonatal sleep patterns and colic frequency. However, this correlation had not been fully explored due to the difficulty of ensuring real melatonin concentration in this complex sample. Given the low concentrations of melatonin and its metabolites in breast milk (1.5-215 pg mL-1), highly sensitive techniques such as liquid chromatography-mass spectrometry (LC-MS) are required. However, the complexity of the matrix usually demands efficient sample preparation techniques, so disposable pipette extraction (DPX) stands out.
    RESULTS: To enhance the selectivity of DPX, molecularly imprinted polymers (MIPs) were applied as the extraction phase due to their ability to create selective binding sites for target molecules. In this study, a DPX-MIP/LC-MS method was developed to determine melatonin and its metabolites in breast milk. To this aim, different monomers were evaluated for the synthesis of MIP, and scrutinized for improved MIP selectivity, where 1-vinylmizadol (1-Vn) presented higher extraction efficiency and was selected as DPX extraction phase. The DPX-MIP method was optimized using both univariate and multivariate approaches. The optimized method exhibited good linearity, minimal matrix interference, recoveries varying from 87.0 % to 111.2 %, and precision between 2.9 % and 14.0 %.
    SIGNIFICANCE: By incorporating MIPs into a miniaturized and automated format, the DPX-MIP/LC-MS method provides a rapid, selective, and sustainable solution for analyzing trace levels of melatonin in breast milk. This approach reduces sample preparation time and enhances selectivity and sensitivity in LC-MS analysis. Overall, the method highlights the potential of MIP-based sample preparation as a powerful strategy for trace-level determination in complex biological matrices.
    Keywords:  AFMK; AMK; Analytical method validation; Disposable pipette extraction; Melatonin; Miniaturized sample preparation technique; Molecularly imprinted polymer
    DOI:  https://doi.org/10.1016/j.aca.2025.344369
  43. Sci Rep. 2025 Aug 01. 15(1): 28183
      Multimodal image registration plays a crucial role in biomedical research, enabling the integration of complementary information from different imaging techniques. We present a novel feature-based approach for multimodal image registration, alongside traditional intensity-based methods. Our method, inspired by SPP-net architecture, employs multi-level feature extraction for robust image alignment. Additionally, we perform t-SNE dimensionality reduction on the MALDI-MSI dataset to enhance feature discrimination and visualization. We evaluated both approaches using datasets from the ANHIR Grand Challenge and mass spectrometry imaging modalities (LA-ICP-MS and MALDI-MSI). The proposed feature-based method achieved comparable accuracy to optimized intensity-based approaches, with Dice Coefficients of 0.95 for ANHIR samples (e.g., COAD_05) and 0.97 for mass spectrometry data, while requiring approximately 50% less computational time. Quantitative evaluation through Mutual Information metrics and Hausdorff Distance demonstrated high registration accuracy across different tissue types and imaging modalities. These results establish our feature-based approach as an efficient alternative to traditional intensity-based methods for multimodal image registration in biomedical applications.
    DOI:  https://doi.org/10.1038/s41598-025-12862-2
  44. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jul 20. pii: S1570-0232(25)00293-4. [Epub ahead of print]1264 124739
      With the use of newer and more robust antiretrovirals, the risk of viral transmission through breastfeeding has sharply diminished. This has led to a change in guideline recommendations. Currently, breastfeeding is regarded as an equipoise of formula, in case of a well-controlled HIV infection. However, it is not yet fully established whether infant exposure to antiretroviral drugs through breastmilk is a reason for concern. Only sparse and heterogenous data exist on concentrations of dolutegravir, raltegravir, bictegravir and doravirine in breastmilk. So further research is required and to accurately describe infant exposure of antiretrovirals through breastmilk, reproducible bioanalytical methods are needed. Our existing UPLC-MS/MS method for several anti-HIV drugs in EDTA-plasma was modified for validation of dolutegravir, bictegravir, raltegravir and doravirine concentrations in human breastmilk. [13C,2H5]-Dolutegravir, [13C,2H2,15N]-bictegravir, [2H6]-raltegravir and [13C6]-doravirine were used as internal standard. The sample preparation involved protein precipitation and detection was performed with tandem mass spectrometry (MS/MS) in a total runtime of 10 min. The assay was validated over the concentration range of 0.0100-10.0 mg/L for dolutegravir and doravirine, 0.00500-10.0 mg/L for raltegravir and 0.0200-20.0 mg/L for bictegravir. Within-run and between-run accuracy were within ±10 % of the nominal concentration and precision <10 CV% for quality controls at high, medium and low concentrations, and ± 8.8 % and < 14 CV%, respectively, at the lower limit of quantification for all analytes. Extraction recovery was 81 % and 79 % for dolutegravir and its internal standard, 83 % and 83 % for bictegravir and its internal standard, 89 % and 92 % for raltegravir and its internal standard and 104 % and 101 % for doravirine and its internal standard. Processed samples of dolutegravir, bictegravir, raltegravir and doravirine in breastmilk were 108 %, 95.5 %, 105 % and 102 % after 14 days at 4 °C. The assay is currently being implemented successfully in pharmacokinetic studies.
    Keywords:  Bictegravir; Breastmilk; Dolutegravir; Doravirine; MS/MS detection; Raltegravir; Ultra-high performance liquid chromatography
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124739
  45. Int J Environ Res Public Health. 2025 Jul 17. pii: 1130. [Epub ahead of print]22(7):
      This study presents an advanced analytical method for the simultaneous quantification of malondialdehyde (MDA), a biomarker of oxidative stress, and diphenyl phosphate (DPhP), a metabolite of the organophosphate flame retardant triphenyl phosphate (TPhP), in human urine. The method integrates hydrophilic interaction liquid chromatography (HILIC), a type of liquid chromatography suitable for polar compounds, for MDA separation, and an online restricted access material (RAM), a preconcentration column, for DPhP isolation, achieving high specificity and sensitivity. Validation with certified urine samples confirmed its robustness across diverse analyte concentrations and complex biological matrices. The optimized clean-up steps effectively minimized carryover, allowing for high-throughput analysis. Application to 72 urine samples revealed a significant positive correlation (ρ = 0.702, p-value = 1.9 × 10-7) between MDA and DPhP levels, supporting a potential link between oxidative stress and TPhP exposure. The subset analysis demonstrated a statistically significant moderate positive correlation in women (ρ = 0.622, p-value = 0.020), although this result should be interpreted with caution because of the limited sample size (N = 14). This method provides a powerful tool for biomonitoring oxidative stress and environmental contaminants, offering valuable insights into exposure-related health risks.
    Keywords:  hydrophilic interaction liquid chromatography; mass spectrometry; organophosphate flame retardants; oxidative stress; restricted access material
    DOI:  https://doi.org/10.3390/ijerph22071130
  46. ACS Chem Neurosci. 2025 Jul 31.
      Alzheimer's disease (AD), characterized by β-amyloid plaques, is increasingly recognized by lipid dysregulation as a key factor in its pathology. Mass spectrometry imaging (MSI), a powerful tool for mapping the spatial distribution of biomolecules in tissue sections, is ideally suited for investigating region-specific molecular alterations in diseased animal tissues. Recent MSI advancements have revealed plaque-associated molecular features in the AD brain, highlighting the role of metabolic dysfunction in disease progression. In this study, we developed a novel multimodal MSI approach using nanospray desorption electrospray ionization (nano-DESI) for dual polarity mode lipid and peptide imaging in the brain tissues of 5-7-month-old transgenic familial AD mice (5xFAD), followed by fluorescence imaging of β-amyloid plaques on the same tissue section. Our results revealed the accumulation of several peptides and phospholipids, including phosphatidylethanolamines (PE), phosphatidylinositols (PI), phosphatidylglycerols (PG), and phosphatidylcholines (PC) in and/or surrounding β-amyloid plaques in the hippocampus, isocortex, and thalamus regions of the AD brain. Furthermore, we observed that several fatty acids (FAs) were enhanced in the plaque-enriched subiculum region of the hippocampus. Our results demonstrate the power of the multimodal nano-DESI MSI approach for comprehensive mapping of molecular pathology with high spatial resolution, providing unique insights into disease metabolism and potential biomarkers.
    Keywords:  Alzheimer’s disease; amyloid plaques; mass spectrometry imaging; multimodal imaging; nano-DESI; spatial lipidomics
    DOI:  https://doi.org/10.1021/acschemneuro.5c00144
  47. Anal Chim Acta. 2025 Oct 08. pii: S0003-2670(25)00767-6. [Epub ahead of print]1370 344373
       BACKGROUND: Conventional sampling methods for clinical analysis make use of plasma or serum and require large blood volumes, cold storage, and specialized handling. This renders them impractical and invasive, especially when dealing with vulnerable groups of patients and the analysis of low-abundant and unstable compounds such as oxylipins. Dried blood micro-sampling methods are designed for collecting minimally invasive small-volume blood samples (<100 μL), providing advantages in stability, handling, and storage. The aim of this study is to develop and compare micro-sampling approaches that can be readily implemented in clinical analysis circumventing the barriers of conventional sampling methods. (96) RESULTS: This study assessed the performance of Pre-Cut Dried Blood Spots (PCDBS) and Volumetric Absorptive Micro-Sampling (VAMS®) devices for determining oxylipins, using small-volume liquid whole blood sampling as reference. A Liquid Chromatography - tandem Mass Spectrometry method was successfully developed and validated. Both PCDBS and VAMS® were prepared using 30 μL of spiked blood and dried for 2 h before extraction and analysis. Optimal results were achieved by pre-treating devices with antioxidant before sample deposition and avoiding the internal standard to undergo evaporation steps during preparation of micro-sampling devices. Long-term storage effects were assessed over a three-month period, and the micro-sampling devices were tested on umbilical cord blood samples from 35 newborns. For PCDBS and VAMS®, eight and nine oxylipins, respectively, showed comparable results to those obtained from liquid whole blood samples with mean concentrations ranging between 0.7 and 6.3 nM (140) SIGNIFICANCE: This study is the first to analyse oxylipins in umbilical cord blood using VAMS® and PCDBS micro-sampling devices, demonstrating their effectiveness for quantification at nM concentrations. It highlights the impact of sample collection, storage, and internal standard handling on lipid profiles. Stability is critical and cold storage (-20 °C) is mandatory. Future research focusing on the standardization of protocols for comparability among laboratories and enhancing sensitivity with improved LC-MS/MS techniques to expand accessible oxylipins are needed. (75).
    Keywords:  Dried blood spot; Inflammation biomarkers; Micro-extraction; Oxylipins; Ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry; Volumetric absorptive Micro-Sampling®
    DOI:  https://doi.org/10.1016/j.aca.2025.344373
  48. Foods. 2025 Jul 09. pii: 2421. [Epub ahead of print]14(14):
      A sensitive method was developed for detecting diazepam residues in aquatic products using magnetic dispersive solid-phase extraction (MDSPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples extracted with 1% ammonia-acetonitrile were purified using synthesized Fe3O4@SiO2-PSA nanoparticles via MDSPE before UPLC-MS/MS analysis. Separation was performed on a C18 column with gradient elution using 0.1% formic acid-2 mM ammonium acetate/methanol. Detection employed positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. Characterization confirmed Fe3O4@SiO2-PSA's mesoporous structure with excellent adsorption capacity and magnetic properties. The method showed good linearity (0.1-10 μg/L, r > 0.99) with an LOD and LOQ of 0.20 μg/kg and 0.50 μg/kg, respectively. Recoveries at 0.5-15.0 µg/kg spiking levels were 74.9-109% (RSDs 1.24-11.6%). This approach provides rapid, accurate, and high-precision analysis of diazepam in aquatic products, meeting regulatory requirements.
    Keywords:  UPLC-MS/MS; aquatic products; benzodiazepine; food safety; veterinary drugs
    DOI:  https://doi.org/10.3390/foods14142421
  49. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2025 Aug 01. 1-21
      Aminoglycosides (AGs) are broad-spectrum bactericidal antibiotics commonly used in animal husbandry. Residues of AGs in food can enter the human body through the food chain and biomagnification. Due to the specific structure and properties of AGs, it is difficult to achieve adequate extraction and separation during analysis, often giving rise to matrix effects. An LC-MS/MS analysis after cleanup based on dipole interaction, hydrogen bonding, adsorption and ion interaction, was developed to determine 16 AGs residues in livestock and poultry meat. The samples were extracted with phosphate buffer (pH 4.0, containing trichloroacetic acid, EDTA-2Na, 0.05 moL L-1 sodium pentanesulfonate, and sodium hexanesulfonate), purified by HLB solid-phase extraction column, and retained and separated by HILIC column with hydrophilic interaction. High correlation coefficients (R2 > 0.99) of calibration curves for the analytes were obtained, linear from 25 to 1500 ng mL-1. The detection limits were 0.006-1.0 μg kg-1, the limits of quantification were 0.02-3.0 μg kg-1. Reasonable recoveries (61%-119%) were demonstrated with acceptable relative standard deviation (RSD < 18%) at 50, 250, and 500 μg kg-1. The signals due to most AGs were affected by different strong matrix effects of livestock and poultry meat, and showed different inhibition and enhancement phenomena, resulting in false negative/positive misjudgments in the results. In terms of overall data and consideration, this method should be an efficient approach for multi-residue analysis as a reliable, selective, and highly sensitive methodology for the simultaneous quantification and confirmation of AGs in livestock and poultry meat.
    Keywords:  Aminoglycosides (AGs); ion pair multiple level interaction; liquid chromatography-tandem mass spectrometry (LC-MS/MS); livestock and poultry meat; solid-phase extraction (SPE)
    DOI:  https://doi.org/10.1080/19440049.2025.2537865
  50. J Adv Res. 2025 Jul 30. pii: S2090-1232(25)00581-8. [Epub ahead of print]
       INTRODUCTION: Metabolic reprogramming plays a significant role in the emergence, progression, and response to antibiotic pressure in bacterial resistance. Current metabolomics approaches face significant limitations: untargeted methods lack quantitative precision, while targeted analyses suffer from limited coverage. These technical constraints hinder comprehensive evaluation of metabolic contributions to antibiotic activity and resistance evolution, creating a critical knowledge gap in understanding treatment outcomes for resistant bacteria.
    OBJECTIVE: To establish a metabolomics method with comprehensive coverage, excellent reproducibility, high sensitivity, and wide dynamic range for elucidating the dynamic relationships between bacterial metabolic reprogramming, antibiotic activity, and resistance phenotype development.
    METHODS: We employed a complementary liquid chromatography system incorporating reverse-phase liquid chromatography, hydrophilic interaction liquid chromatography, and metal-sensitive liquid chromatography. Coupled with high-resolution mass spectrometry and utilizing three complementary data acquisition modes - full scan, information-dependent acquisition (IDA), and sequential window acquisition of all theoretical mass spectra (SWATH) - we developed a novel pseudo-targeted metabolomics approach based on triple quadrupole mass spectrometry, designated as SWATH/IDA-MRM. This optimized method was subsequently applied to investigate metabolic reprogramming in Escherichia coli strains harboring the resistance genes mcr-1, blaNDM-1, blaNDM-5, and the dual combination mcr-1 + blaNDM-1.
    RESULTS: Our analytical platform successfully identified 3,529 metabolic features using six complementary chromatographic separation conditions, achieving broader metabolite coverage than conventional targeted metabolomics. Comparative evaluation against untargeted approaches revealed marked improvements in analytical performance, including enhanced linearity, reproducibility, detection sensitivity, and dynamic range, along with superior capacity for discriminating metabolic profiles between sample groups. Application to antibiotic-resistant E. coli strains revealed substantial metabolic flux alterations in resistant versus susceptible strains, with predominant perturbations in nucleotide metabolism, amino acid metabolism, energy metabolism, lipid metabolism, and redox balance pathways.
    CONCLUSION: The developed SWATH/IDA-MRM platform represents a significant methodological advancement for investigating the complex interplay between microbial metabolic adaptation and antimicrobial responses. This integrated analytical approach enables systematic characterization of resistance-associated metabolic reprogramming, thereby establishing a framework for developing targeted therapeutic strategies against pathogen-specific metabolic vulnerabilities.
    Keywords:  Bacterial metabolism; Bla(NDM); Pseudo-targeted metabolomics; mcr-1
    DOI:  https://doi.org/10.1016/j.jare.2025.07.051
  51. PLoS One. 2025 ;20(7): e0329115
      In marine ecosystems, microbial communities often interact using specialised metabolites, which play a central role in shaping the dynamics of the ecological networks and maintaining the balance of the ecosystem. With metabolomics and transcriptomics analyses, this study explores the interactions between two marine microalgae, Skeletonema marinoi and Prymnesium parvum, grown in mono-cultures and non-contact co-cultures. As a growth indicator, the photosynthetic potential, measured via fluorescence, suggested chemical interaction between S. marinoi and P. parvum. Using Liquid Chromatography-Mass Spectrometry (LC-MS) data, we identified 346 and 521 differentially produced features in the endo- and exometabolome of S. marinoi and P. parvum, respectively. Despite limited tandem mass spectrometry data (MS2) for these features, we structurally annotated 14 compounds, most of which were previously under-studied specialised metabolites. Differential gene expression analysis was then performed on the transcriptomes of the microalgae, which uncovered differentially expressed genes involved in energy metabolism and cellular repair for both species. These metabolic changes depict the adaptation of both species in the co-culture. However, further data acquisition and investigation will be necessary to confirm the type of interaction and the underlying mechanisms.
    DOI:  https://doi.org/10.1371/journal.pone.0329115
  52. J Sci Food Agric. 2025 Jul 29.
       BACKGROUND: The global rise in antibiotic use, particularly tetracyclines (TCs) in animal husbandry, poses significant risks to food safety and public health due to residual accumulation and bacterial resistance. Existing detection methods for TCs in dairy products often lack efficiency, sensitivity, or the capability for simultaneous multi-residue analysis. Developing a rapid, precise, and cost-effective method to monitor TCs in compliance with international maximum residue limits remains an urgent need.
    RESULTS: We developed a streamlined approach integrating a three-step purification process (PRiME HLB cartridges) with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method utilizes 0.1 mol L-1 Na2EDTA-McIlvaine buffer for extraction, achieving 83.11-107.61% recovery rates with < 5.43% relative standard deviation (RSD). Optimized chromatographic separation on a bridged ethyl-siloxane and silica hybrid (BEH) C18 column enabled simultaneous detection of 11 TCs (including parent compounds and metabolites) within 9 min. Sensitivity was exceptional, with limits of detection and limits of quantification of 0.02-0.83 μg kg-1 and 0.07-2.78 μg kg-1, respectively. Intra-/inter-day precision (RSD 3.03-9.33%) and compliance with European Union/Food and Drug Administration (EU/FDA) were validated using real milk and milk powder samples.
    CONCLUSIONS: This method combines rapid sample preparation with UPLC-MS/MS for simultaneous quantification of 11 TCs in dairy products. Its high accuracy, sensitivity, and compliance with global regulatory standards make it indispensable for large-scale food safety monitoring, effectively addressing antibiotic residue risks and supporting international trade harmonization. © 2025 Society of Chemical Industry.
    Keywords:  UPLC–MS/MS; milk safety; multi‐residue analysis; tetracycline antibiotics; veterinary drug residues
    DOI:  https://doi.org/10.1002/jsfa.70088
  53. Rapid Commun Mass Spectrom. 2025 Nov 15. 39(21): e10111
       RATIONALE: Novel psychoactive substances, especially those within the benzimidazole class such as metonitazene, are powerful psychoactive agents with considerable potential for misuse in both human and animal sports. Implementing a robust detection and monitoring system is crucial to ensure fair competition and welfare of the athletes involved.
    METHODS: In vitro studies were conducted using camel liver homogenates to investigate the metabolism of metonitazene in camels. The metabolites were analyzed using a Thermo Fisher Orbitrap Exploris LC-MS system. Method validation for qualitative determination was performed using in-house developed methods, while data analysis and metabolite identification were performed using the Compound Discoverer software.
    RESULTS: A total of seven Phase I metabolites of metonitazene were successfully identified. The metabolic transformations were predominantly characterized by dealkylation reactions. These metabolites hold promise as potential markers for the long-term detection of metonitazene in camels for doping control applications.
    CONCLUSION: This study highlights the effectiveness of advanced high-resolution LC-MS techniques in identifying and characterizing the in vitro metabolites of metonitazene in camels. Considering the high potency and potential for abuse of metonitazene in camel racing, the metabolites identified offer a valuable basis for establishing robust doping control strategies. These results support the development of regulatory frameworks designed to protect animal welfare and uphold the integrity of the sport.
    Keywords:  LC‐HRMS; camel racing; doping control; metabolism; metonitazene; synthetic opioids
    DOI:  https://doi.org/10.1002/rcm.10111
  54. Rapid Commun Mass Spectrom. 2025 Nov 30. 39(22): e10112
       RATIONALE: Atmospheric solids analysis probe-mass spectrometry (ASAP-MS) is an established ambient ionisation technique that allows for the direct and rapid analysis of samples without chromatographic separation. Consequently, applications that typically benefit from ambient ionisation approaches can achieve improved sample throughput and thus improved potential for in situ testing. Previous reports have contrasted in viewpoints on the ability for ASAP-MS to provide reliable quantitative data. Critically, in-depth data exploring the quantitative capabilities of ASAP-MS are currently lacking.
    METHODS: Here, a series of experiments were performed to assess the quantitative performance of ASAP-MS using a proof-of-concept single analyte (caffeine) approach. Analytical precision, accuracy, linearity and sensitivity were investigated using numerous variables, including sample deposition method (i.e., directly placing the probe into the sample vs. pipetting the sample onto the probe) and deposition volume, as well as the presence of a series of different internal standard approaches.
    RESULTS: The data acquired demonstrated that the use of a positive displacement pipette and an isotopically labelled (structure-matched) internal standard provided an optimal approach for quantitative reliability, albeit at levels often below the standards set for traditional chromatography-based quantitative assays. The investigations were performed across a concentration range of 50-5000 ng/mL. Whereas measurable responses were seen across the full range in most approaches, limitations in sensitivity were identified and reduced quantitative performance statistics were noted at concentrations below 1000 ng/mL.
    CONCLUSIONS: These experiments demonstrate, for the first time, a comprehensive investigation into the quantitative performance of ASAP-MS using caffeine as the example analyte. These data offer insight into the current strengths and limitations of quantitative analyses using ASAP-MS and aim to provide practical recommendations to optimise quantitative approaches using this technique.
    Keywords:  ASAP‐MS; accuracy; ambient ionisation mass spectrometry; precision; quantitation
    DOI:  https://doi.org/10.1002/rcm.10112
  55. Rapid Commun Mass Spectrom. 2025 Nov 15. 39(21): e10114
       RATIONALE: This work introduces an alternative experimental approach by integrating high-resolution mass spectrometry (HRMS) with multivariate statistical analysis for metabolite detection and identification. The integration of these tools maximizes information extraction from data, improving accuracy and reducing the risk of false identifications.
    METHODS: Seven volunteers' urine samples were collected before and after oral administration of 10 mg of methylclostebol (4-chloro-17β-hydroxy-17α-methylandrost-4-en-3-one, ClMT) and assigned to three excretion time intervals. Analyses were carried out on a GC-HRMS system (Agilent 8890 GC coupled with 7250 GC/QTOF), utilizing low-energy electron ionization (< 18 eV) to preserve the native molecular skeleton, thereby simplifying mass spectrum interpretation, with acquisition in full scan mode. Raw data were then processed and subjected to multivariate analysis.
    RESULTS: The orthogonal partial least squares-discriminant analysis (OPLS-DA) was employed to emphasize differences among specific sample conditions, and features that significantly contribute to classification in the OPLS-DA can be identified as important biomarkers. Samples from the three excretion intervals demonstrated clear separations, occupying distinct areas within the model's defined space. From this approach, the S-plot displayed seven features identified as biomarkers related to methylclostebol ingestion, comparing their mass spectra with an in-house library of LE-EI mass spectra.
    CONCLUSIONS: The application of this approach is demonstrated to enhance the identification of new markers related to the intake of prohibited substances in the anti-doping field, such as methylclostebol. Its application proved to be an alternative strategy that allows for gathering a more comprehensive range of information in the antidoping field.
    Keywords:  OPLS‐DA; mass spectrometry; metabolomics; methylclostebol; untargeted
    DOI:  https://doi.org/10.1002/rcm.10114
  56. Food Chem. 2025 Jul 16. pii: S0308-8146(25)02793-1. [Epub ahead of print]492(Pt 3): 145542
      Human milk is a crucial source of thyroid hormones, essential for neonatal development. We developed and validated a three-phase hollow-fiber liquid-phase microextraction method, coupled to ultra-high performance liquid chromatography and ion mobility mass spectrometry, to simultaneously quantify eight thyroid hormones, including the chiral forms D-thyroxine and L-thyroxine in human milk. The method showed excellent linearity (R2 > 0.999), low limits of detection (0.7-19.8 μg L-1), and high recoveries (83-114 %). The analysis of 30 human milk samples, revealed that D-thyroxine (115.1-157.5 μg L-1) was more abundant than L-thyroxine (47.9-193.6 μg L-1), and reversed triiodothyronine prevailed over triiodothyronine. This is the first report of chiral forms of thyroxine, diiodotirosine, and thyronine in human milk, with potential implications for lipid metabolism and infant thyroid regulation. These findings highlight the method sensitivity and biological relevance, offering a robust tool for future nutritional and endocrine studies.
    Keywords:  Hollow-fiber liquid-phase microextraction; Human milk; Iodine speciation; Ion mobility; Quadrupole time of flight; Thyroid hormones; Ultra-high-performance liquid chromatography
    DOI:  https://doi.org/10.1016/j.foodchem.2025.145542
  57. Anal Chem. 2025 Aug 01.
      Retention time (RT) can provide orthogonal information to mass spectra, supporting the qualitative identification. However, RT is influenced by experimental conditions and column parameters, and it is difficult to have a large amount of RT data in the user's experimental conditions. Hence, various machine learning methods, including advanced deep learning approaches, have been developed for RT prediction. However, most of them were limited to a given column and operational conditions. In the meantime, data sparsity often hinders the prediction performance. In this study, we propose an MDL-TL method that combines multiple data sets to jointly train the base model. MDL-TL vectorizes the column and conditions (chromatographic parameters, CPs) using word2vec and autoencoders, and distinguishes the data sets from different chromatographic experiments by including the CPs in the compound representation. This not only augments the data but also introduces the CPs into the RT prediction, allowing the pretrained model to be efficiently transferred to different target systems by fine-tuning. MDL-TL was evaluated against five popular deep learning approaches and four machine learning approaches on 14 reversed-phase liquid chromatography data sets and 14 hydrophilic interaction liquid chromatography data sets, respectively. The results show that our method surpassed the compared methods, including transfer learning methods based on the METLIN small molecule retention time (SMRT) data set, in mean absolute error, median absolute error, mean relative error, and R2 in most cases, demonstrating that MDL-TL is a promising approach for predicting RTs for various chromatographic systems and operational conditions.
    DOI:  https://doi.org/10.1021/acs.analchem.5c01703
  58. J Addict Med. 2025 Jul-Aug 01;19(4):19(4): 484-486
       OBJECTIVE: Herein, we evaluate the performance of xylazine test strips (XTS) in urine samples. XTS is used for community drug checking (powders and liquids) but lacks regulatory approval for human specimen testing.
    METHODS: We obtained n=85 human urine specimens from a toxicology laboratory in Philadelphia, originally submitted for qualitative mass spectrometry (MS) expanded drug analysis. Residual urine was tested for xylazine using XTS (BTNX Inc.), and results were then compared against the MS method. Synthetic urine spiked with xylazine standards was used to determine the XTS cutoff. An external quantitative MS method was used to investigate potential mismatches.
    RESULTS: Of n = 85 human urine specimens, XTS demonstrated 86% sensitivity and 93% specificity using a XTS cutoff of 750 ng/mL established with synthetic urine samples. Six false negatives (14%) among 43 qualitative MS-positive samples were observed, primarily due to XTS's lower sensitivity. Among 3 false positives (7%) observed in 42 qualitative MS-negative samples, lidocaine likely causes the interference. Interestingly, some XTS-positive samples were found to have xylazine concentration lower than 750 ng/mL using quantitative MS, suggesting cross-reactivity with unknown metabolites or analogs.
    CONCLUSIONS: XTS requires further refinement to achieve lab-quality performance, with a focus on improving sensitivity and minimizing false positives caused by nonspecific interactions with urine components. Further research is necessary to optimize their design, establish accurate detection thresholds, supporting clinical decision-making, and obtain regulatory validation.
    Keywords:  adrenergic alpha-2 receptor agonists; fentanyl; hypnotics and sedatives; xylazine
    DOI:  https://doi.org/10.1097/ADM.0000000000001497
  59. J Pharm Biomed Anal. 2025 Jul 24. pii: S0731-7085(25)00422-4. [Epub ahead of print]266 117081
      Understanding the structural diversity and biological functions of unsaturated fatty acyl chains (FAC) esterified in complex lipids -such as glycerolipids (GL), glycerophospholipids (GP) or sphingolipids (SP)- requires precise knowledge of the degree of unsaturation, location, and geometrical isomerism of the carbon-carbon double bonds (CC). However, the complex isomeric nature of lipids, combined with the inherent limitations of conventional tandem mass spectrometry (MS/MS) in structural elucidation, remains a major challenge in accurate CC elucidation. To overcome this, advanced MS/MS strategies, such as electron impact excitation of ions from organics (EIEIO) have emerged, generating diagnostic fragment ions that enable unambiguous CC localization. In the present study, we conducted a qualitative structural analysis of the CC positions in esterified FAC of GP present in NIST® Human Plasma Standard Reference Material, SRM 1950, employing RP-UHPLC-ESI(+)-EIEIO-QTOF-MS/MS. Interpretation of ESI(+)-EIEIO-MS/MS spectra enabled CC determination in 120 unsaturated GP, revealing a predominance of ω-6 and ω-3 FAC. These results offer new insights into the FAC distribution of this reference material, enhancing the structural annotation confidence level. By integrating such detailed molecular information, EIEIO-MS/MS proves to be a powerful approach for in-depth lipid structural elucidation in complex biological matrices, thereby contributing to methodological advancements and supporting its future application in translational lipidomics.
    Keywords:  Double bond position; Electron impact excitation of ions from organics (EIEIO); Electron-induced dissociation (EID); Lipid annotation; Lipidomics; MS/MS fragmentation; NIST SRM 1950
    DOI:  https://doi.org/10.1016/j.jpba.2025.117081
  60. BMC Microbiol. 2025 Jul 25. 25(1): 452
      Annually, millions of people are affected by mosquito-borne Orthoflavivirus infections. These include diseases caused by the Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), posing a formidable challenge to global public health. This research aims to explore the potential role of the Gut-Brain Axis (GBA) in Orthoflavivirus infection, particularly focusing on key metabolites involved in the process of viral invasion into the central nervous system. Given the advantages of metabolomics technology in metabolite identification. Therefore, we employed an untargeted Liquid Chromatography-Mass Spectrometry (LC-MS) metabolomics platform to examine alterations in metabolite concentrations within the feces and brain tissues of mice infected with DENV, JEV, or ZIKV, as well as uninfected controls. The results showed that 225, 240, and 252 differential metabolites were identified in the fecal metabolome of DENV, JEV, and ZIKV infections, respectively, with amino acid metabolism and lipid metabolism being significantly disrupted. In the brain metabolome, 37, 81, and 18 differential metabolites were identified for DENV, JEV, and ZIKV infections, respectively, with lipid metabolism and purine metabolism being significantly disrupted. Amino acids with low abundance in viral proteins are significantly disrupted in the amino acid metabolism pathway, suggesting that Orthoflaviviruses adapt to its needs for synthesizing viral proteins by regulating the host's amino acid composition. The disruption of purine metabolism also implies the viral genome replication process occurring in the brain. Moreover, the disturbance of lipid metabolism is highly correlated with the biological function of the Orthoflavivirus envelope, where Sphingosine 1-phosphate (S1P) may be the key for Orthoflaviviruses to enter the human central nervous system via the GBA. This research is the first to explore the potential role of GBA in Orthoflavivirus infection through joint metabolomic analysis of fecal and brain tissue samples, providing new insights into viral invasion of the central nervous system. The findings not only elucidate the characteristics of viral infection from complementary perspectives of fecal and brain tissue samples, revealing associated metabolic changes, but also establish a foundation for subsequent identification of biomarkers to diagnose disease states-particularly for predicting central nervous system infection risks. The specific patterns revealed by fecal metabolomics analysis provide the theoretical basis for developing non-invasive predictive approaches to assess brain infection status in the future.
    Keywords:   Orthoflavivirus ; Biomarkers; GBA; LC-MS; Metabolomics
    DOI:  https://doi.org/10.1186/s12866-025-04192-0
  61. J Chromatogr A. 2025 Jul 16. pii: S0021-9673(25)00574-6. [Epub ahead of print]1759 466229
      Miniaturized sample preparation methods are becoming a growing trend in analytical chemistry to reduce solvent/ sample consumption and enhance analytical performance. This study encompasses evaluating the performance of microwave-assisted extraction and ultrasound-assisted extraction for extraction of favipiravir from dried plasma spots after on-spot protein denaturation. Each extraction method was combined with high-performance liquid chromatography with UV detection. Microwave-assisted extraction was performed using methanol (10 µL) as an on-spot plasma protein denaturant, the mobile phase as extraction solvent, and microwave power of 50 % for 40 s. Ultrasound-assisted extraction was accomplished using ethanol (10 µL) as a denaturant and mobile phase as a solvent of extraction for 40 min. The developed methods were validated as per the International Council for Harmonization guidelines M10. Both methods showed very good linearity in the concentration range of 10-50 μg mL-1, negligible matrix effect and carryover, and good selectivity and specificity. The recovery results were found to be between 87 - 99 % and 93 - 103 %, for microwave and ultrasound methods, respectively. The methods were successfully applied for favipiravir determination in real human plasma. An incurred real sample reanalysis study was performed to investigate dried sample stability, revealing satisfactory sample stability for up to one month in both freezer and refrigerator as indicated by % differences <13 %. The comparative analysis of methods' profiles, using different greenness and whiteness metric tools, revealed superior greenness and analytical performance of ultrasound-assisted extraction with similar practicality. The developed methods exhibited excellent analytical performance, increased throughput, reduced volumes of sample (10 μL) and organic solvents (210 μL), and negligible waste. Thus, it can be adopted for routine work in pharmacokinetics and clinical trial studies.
    Keywords:  Dried plasma samples; Favipiravir; High-throughput; Microwave-assisted extraction; Miniaturization; Ultrasound-assisted extraction
    DOI:  https://doi.org/10.1016/j.chroma.2025.466229