bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2026–04–19
forty-four papers selected by
Sofia Costa, Matterworks
  1. A rapid 2-minute LC-MS/MS method with simple protein precipitation for simultaneous determination of tryptophan and its two metabolites kynurenine and kynurenic acid in human serum.
  2. Simultaneous quantification of 18 serum steroids and catecholamines via LC-MS/MS: A derivatization-free method using mixed-mode SPE and PFP chromatography.
  3. Simultaneous Determination of Fexinidazole and Its Major Metabolites in Rat Plasma by UPLC-MS/MS: Method Validation and Application to Pharmacokinetic Study.
  4. [Determination of 65 synthetic cannabinoids in whole blood by QuEChERS-ultra performance liquid chromatography- mass spectrometry].
  5. Lipidomic and Metabolomic Profiling on Low-Count Human Spermatozoa: A Robust and Reproducible Method for Untargeted HPLC-ESI-MS/MS-Based Approach.
  6. [Determination of 38 antibiotic residues in honey by magnetic dispersive solid-phase extraction-liquid chromatography-tandem mass spectrometry].
  7. LC-MS/MS Method for Therapeutic Drug Monitoring of Abiraterone, Darolutamide, Apalutamide, Enzalutamide, and Metabolites in Prostate Cancer Patients.
  8. Disentangling method-induced differences in metabolomics: single- versus dual-phase extraction.
  9. Protocol for metabolite extraction and sample preparation from primary astrocytes for mass spectrometry-based metabolomic analysis.
  10. Rapid quantitative bioanalysis using LC-HRIM-QTOFMS with flexible sample delivery and variable traveling wave profiles.
  11. Optimisation and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for cannabis and its metabolites in urine.
  12. [Determination of ergot alkaloids toxins in forage grass by ultra performance liquid chromatography-tandem mass spectrometry coupled with solid-phase supported liquid-liquid extraction].
  13. A High-Throughput LC-MS/MS Bioanalytical Method for Simultaneous Determination of Mirabegron and Solifenacin in Pharmacokinetic Investigations.
  14. A Sensitive and Selective UHPLC-MS/MS Method Based on Mercaptopropionic Acid Derivatization for Quantification of Juglone in Biological Matrices.
  15. [Rapid determination of lubabegron residues in animal-derived foods by ultra performance liquid chromatography- tandem mass spectrometry].
  16. Development of analytical methods for the quantification and enhanced detection of unsaturated short-chain FAHFAs.
  17. A chromatographic approach to avoid the matrix effect caused by sodium acetate clusters in the liquid chromatography coupled with electrospray ionization mass spectrometry analysis of free fatty acids in human plasma.
  18. GC-MS method development and validation for the determination of Short Chain Fatty Acids in human feces.
  19. Establishment and application of a QuEChERS-based high-performance liquid chromatography-mass spectrometry method for the analysis of 56 per- and polyfluoroalkyl substances in human plasma.
  20. Determination of Glycidol in Soy Sauce Using p-Dimethylaminophenol Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry.
  21. Development and validation of an LC-MS/MS assay for serum 5α-androstane-3α, 17β-diol 17-glucuronide with enhanced interference resolution.
  22. Heterologous internal calibration for multiplexed internal quantification in targeted LC-MS/MS bioanalysis.
  23. Pitfalls of onco-metabolomics: impact of sample integrity on metabolomic investigations in more than 4500 human serum samples from ten different cohorts.
  24. Therapeutic oligonucleotide (nusinersen) metabolism in cerebrospinal fluid samples of patients with spinal muscular atrophy based on liquid chromatography coupled with mass spectrometry data.
  25. Development of electromembrane extraction using deep eutectic solvents for 22 representative veterinary drug residues in honey, milk, and eggs.
  26. Comparison of Extraction Methods for the Quantification of Phytohormones from Tomato Fruits and Leaves by LC-MS/MS.
  27. Tri-(2-ethylhexyl) trimellitate - Determination of 1-MEHTM, 2-MEHTM, 5OH-1-MEHTM, 5OH-2-MEHTM, 5cx-1-MEPTM, and 5cx-2-MEPTM in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
  28. [Determination of eight ultraviolet absorbers in surface water and wastewater by ultra performance liquid chromatography-triple quadrupole mass spectrometry].
  29. Mycotoxins - Determination of deoxynivalenol and deepoxydeoxynivalenol in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
  30. Structure-centric searching enables global mapping of the public metabolome.
  31. Rapid Cold Fixation of Mouse Colon (Col'RFix) Enables High-Resolution Mass Spectrometry Imaging.
  32. Decoding the Oxylipin Chemical Space Using Ion Identity Molecular Networking.
  33. Ultrafiltration-MSPE with in-situ quaternary aminooxy oximation for LC-MS/MS quantification of nine serum free androgens.
  34. Mycotoxins - Determination of aflatoxins, ochratoxin A, free ochratoxin α, gliotoxin, citrinin, and dihydrocitrinone in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
  35. Determining EDC exposure: direct analytical methods are essential for accurate analysis of human biospecimens.
  36. Glyphosate - Determination of glyphosate and AMPA in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
  37. Wooden-Tip Electrospray Ionization Mass Spectrometry Combined With Machine Learning for Differentiating Thyroid Tumours.
  38. Uncovering tire wear signatures in urban aerosols: application of ultrahigh-performance liquid chromatography-tandem mass spectrometry using multimode ionization source for the determination of tire wear compounds.
  39. Validation of a quantitative analysis method for tryptophan metabolites in asthmatic mice and bacteria using GC-MS/MS.
  40. Isotope Dilution NanoLC-MS/MS Quantitation of Methylglyoxal DNA-Protein Cross-Links: Formation and Repair in Human Cells.
  41. Comprehensive profiling of alkaloids in Buxus sinica by integrating global natural products social molecular networking with derivatization-assisted characteristic ion filtering.
  42. A New Potential Biomarker for Strychnine Misuse in Human Urine Using Quadrupole-Orbitrap LC-MS/MS.
  43. Collision energy consistency between analyte and internal standard: A critical factor for accurate quantification of steroid hormones by LC-MS/MS.
  44. Simultaneous LC-MS Profiling of Bioactive Ecdysteroids in Nutrient-Dense Plant Sources and Dietary Supplements.
  1. J Pharm Biomed Anal. 2026 Mar 27. pii: S0731-7085(26)00116-0. [Epub ahead of print]277 117448
    A rapid 2-minute LC-MS/MS method with simple protein precipitation for simultaneous determination of tryptophan and its two metabolites kynurenine and kynurenic acid in human serum.
    Hui Yan, Haoyang Lu, Shanqing Huang, Yuqing Li, Hui Xia, Wanting Huang, Dewei Shang, Ming Zhang.
      Dysregulation of the tryptophan (TRP) metabolic pathway is closely linked to the pathophysiology of neuropsychiatric disorders, such as depression. This study aimed to develop and validate a sensitive, rapid, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of TRP and its metabolites, kynurenine (KYN) and kynurenic acid (KYNA), in human serum. Analytes were extracted from 100 µL of serum via simple protein precipitation with acetonitrile. Chromatographic separation was achieved on an Agilent ZORBAX HILIC Plus column (4.6 mm×100 mm, 3.5 µm) using isocratic elution with a mobile phase of methanol: acetonitrile containing 5 mM ammonium formate. Quantification was performed using an electrospray ionization source in positive ion multiple reaction monitoring (MRM) mode, with a total run time of only 2.0 min.The linear ranges were 1-50 µg/mL for TRP, 0.1-5 µg/mL for KYN, and 1-50 ng/mL for KYNA, covering clinically relevant levels. A weighting factor of 1/x² provided the best fit for calibration curves (R² > 0.99). Extraction recoveries ranged from 88.23% to 99.39%, and mean internal standard-normalized matrix effects were 81%-100%. Accuracy and precision values met bioanalytical acceptance criteria. Stability assessments confirmed that samples were stable at -20°C and -80°C for 31 days and through three freeze-thaw cycles. This validated method was successfully applied to analyze serum samples from 103 adolescent patients with first-episode depression.
    Keywords:  High-performance liquid chromatography; Kynurenic acid; Kynurenine; Tandem mass spectrometry (LC-MS/MS); Tryptophan
    DOI:  https://doi.org/10.1016/j.jpba.2026.117448
  2. J Chromatogr A. 2026 Apr 08. pii: S0021-9673(26)00310-9. [Epub ahead of print]1777 466980
    Simultaneous quantification of 18 serum steroids and catecholamines via LC-MS/MS: A derivatization-free method using mixed-mode SPE and PFP chromatography.
    Yingfeng Gao, Yi Zhang, Xin Meng, Ruiwei Xu, Huixia Liu, Haoyang Yao, Jicheng Gong.
      The quantification of serum steroids and catecholamines is essential for the clinical diagnosis of adrenal diseases and tumors. Due to their distinct physicochemical properties and low serum concentrations, existing assays typically quantify these analytes separately using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Simultaneous quantification offers significant advantages, including enhanced diagnostic accuracy and improved sample comparability. Here, we developed a sensitive LC-MS/MS method to simultaneously measure 6 catecholamines, 10 steroids, and 2 other neuroendocrine hormones in a single run. Sample preparation involved a simple one-step solid-phase extraction without derivatization. All analytes were chromatographically separated on an HSS PFP column within 14 minutes. The method exhibited excellent linearity (r2>0.9930) with limits of quantification ranging from 0.0006 to 2 ng/mL. Extraction recoveries and matrix effects ranged from 82.6% to 117.4% and 82.0% to 107.6%, respectively. Intra- and inter-day reproducibility was validated, with coefficients of variation (CVs) between 0.6% and 11.4%. The method was successfully applied to serum samples from healthy volunteers in an exposure-intervention study. Detection rates exceeded 95% for most analytes, with the exception of dihydrotestosterone and dehydroepiandrosterone. The developed method demonstrates improved sensitivity and throughput compared to existing assays, serving as a valuable quantitative tool for large-scale epidemiological studies and clinical practice.
    Keywords:  Catecholamines; LC-MS/MS; Mixed-mode SPE; Neuroendocrine response; Steroids
    DOI:  https://doi.org/10.1016/j.chroma.2026.466980
  3. Drug Des Devel Ther. 2026 ;20 589234
    Simultaneous Determination of Fexinidazole and Its Major Metabolites in Rat Plasma by UPLC-MS/MS: Method Validation and Application to Pharmacokinetic Study.
    Chenchen Qian, Shiqi Jiang, Haoxin Fu, Yuxin Shen, Ruanjuan Zhan, Xia Lan, Chenjian Zhou.
       Introduction: Fexinidazole is an oral antitrypanosomal drug approved for the treatment of human African trypanosomiasis (HAT). Its therapeutic efficacy primarily depends on the in vivo formation of its active metabolites, sulfoxide fexinidazole (M1) and sulfone fexinidazole (M2). Accurate and sensitive quantification of fexinidazole and its major metabolites is essential for pharmacokinetic evaluation and preclinical research. Therefore, a reliable bioanalytical method for their simultaneous determination in plasma is required.
    Methods: An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of fexinidazole, M1, and M2 in rat plasma, using fluconazole as the internal standard (IS). Chromatographic separation was achieved on an Acquity UPLC BEH C18 column with gradient elution consisting of 0.1% formic acid in water and acetonitrile. The method was validated in accordance with bioanalytical guidelines, including assessments of selectivity, linearity, lower limit of quantification (LLOQ), precision, accuracy, recovery, matrix effects, and stability. The validated method was subsequently applied to a pharmacokinetic study in rats.
    Results: The method demonstrated good linearity within the investigated concentration ranges for all analytes. The LLOQs were 1 ng/mL for fexinidazole, 10 ng/mL for M1, and 50 ng/mL for M2. Intra-day and inter-day precision (RSD%) and accuracy (RE%) were within ±15%. Recovery was consistent and reproducible, matrix effects were acceptable, and stability under various conditions met bioanalytical requirements. The method was successfully applied to the pharmacokinetic evaluation of fexinidazole and its metabolites in rats.
    Discussion: The developed UPLC-MS/MS method exhibited satisfactory sensitivity, accuracy, and reproducibility for the quantification of fexinidazole and its active metabolites in rat plasma. This method provides reliable analytical support for pharmacokinetic studies and contributes to the preclinical investigation of fexinidazole.
    Keywords:  UPLC–MS/MS; fexinidazole; pharmacokinetics; rat
    DOI:  https://doi.org/10.2147/DDDT.S589234
  4. Se Pu. 2026 Apr;44(4): 393-402
    [Determination of 65 synthetic cannabinoids in whole blood by QuEChERS-ultra performance liquid chromatography- mass spectrometry].
    Zhang Xi, Yi Ye, Song-Pan Li, Jing Kang, Wei-Qun Liu, Jing Zhang, Dao-Xia Li.
      Synthetic cannabinoids （SCs） have become the most diverse and widely abused class of new psychoactive substances in the world. Blood and urine are classic sample matrices for in vivo toxin analysis， but some SCs have high lipophilicity， and the concentration of parent drugs in urine is extremely low， making them difficult to detect. Metabolites need to be investigated， and SCs with similar structures can produce the same metabolites. Therefore， establishing urine detection methods and interpreting results are relatively complex. In contrast， the detection of synthetic cannabinoid parent drugs in blood is more straightforward， and detecting parent drugs in blood can serve as direct evidence in legal cases. In order to detect the abuse of SCs， a QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method was developed to simultaneously determine 65 SCs in blood. The sample preparation and detection conditions were optimized. Quantification was achieved using an internal standard and matrix-matched calibration curves， enabling rapid screening and quantitative analysis of the 65 SCs in blood. Following protein precipitation with acetonitrile， the blood extract was further extracted and purified using QuEChERS reagents. Chromatographic separation of all 65 SCs was performed on a Waters Acquity UPLC HSS T3 column （100 mm×2.1 mm， 1.8 μm） maintained at 40 ℃. Detection employed dynamic multiple reaction monitoring （dMRM） mode with a mobile phase consisting of 0.1% （volume fraciotn） formic acid aqueous solution and acetonitrile， delivered at a flow rate of 0.25 mL/min. The injection volume was 2 μL. The 65 SCs exhibited good linear relationships in the mass concentration range of 0.05-200 ng/mL with correlation coefficents （r） exceeding 0.992. The limits of detection （LODs） were between 0.01 and 0.2 ng/mL， and the limits of quantitation （LOQs） were between 0.05 and 0.5 ng/mL， respectively， which meet the requirements for analyzing SCs in blood sample. The intra-day and inter-day precisions （n=6） were determined to be 1.0%-9.9% by spiking blank blood samples with the 65 SCs at mass concentrations of 1， 5， and 50 ng/mL. The recoveries of the 65 SCs were between 62.2% and 116.9%， and the matrix effects were between 70.2% and 117.7%. All analytes demonstrated good stability and acceptable dilution integrity in blood samples. Using the established method， 10 blood samples from suspected drug use cases were successfully screened for SCs. The target compounds were detected in all 10 samples， specifically including ADB-BUTINACA， MDMB-4en-PINACA， MDMB-FUBICA， and 5F-MDMB-PICA with mass concentrations ranging from 1.9 to 23.1 ng/mL. The detection rates of ADB-BUTINACA and MDMB-4en-PINACA were 90% and 50%， respectively， suggesting their relatively high prevalence in China's illegal drug market. In addition， two or more SCs were detected simultaneously in six blood samples. Compared with other existing literature methods， this study combines QuEChERS with precipitation protein method for blood sample pretreatment， greatly improving the detection efficiency and suitable for rapid screening of whole blood samples in batches. The results demonstrate that the established method offers accuracy， rapidity， sensitivity， and effective chromatographic separation. It can serve as a reliable tool for forensic laboratories to perform rapid screening and quantitative analysis of SCs in blood， providing robust technical support for combating drug-related crime and supporting social stability.
    Keywords:  QuEChERS; dynamic multiple reaction monitoring （dMRM）; protein precipitation; synthetic cannabinoids; ultra performance liquid chromatography-mass spectrometry （UPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.04037
  5. Cells. 2026 Apr 05. pii: 649. [Epub ahead of print]15(7):
    Lipidomic and Metabolomic Profiling on Low-Count Human Spermatozoa: A Robust and Reproducible Method for Untargeted HPLC-ESI-MS/MS-Based Approach.
    Irune Calzado, Manu Araolaza, Mikel Albizuri, Ainize Odriozola, Iraia Muñoa-Hoyos, Iratxe Ajuria-Morentin, Nerea Subirán.
      Human infertility affects approximately 17.5% of the global population, with male factors accounting for nearly half of all cases. Identifying reliable molecular biomarkers is crucial for improving the diagnosis and assessment of male fertility. This study established and refined an untargeted high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) protocol for a comprehensive lipidomic and metabolomic analysis of human spermatozoa, using only 1.25 million cells per sample. Compared with previous reports, our optimized method achieved an unparalleled level of analytical depth, identifying 473 lipid species and 955 structurally annotated metabolites. This corresponds to nearly a 7600-fold improvement in detection efficiency per cell compared with previously published approaches. Lipidomic analysis revealed that the most abundant lipid classes were glycerophospholipids (39%), cholesterol (20%) and fatty acids (19%), with cholesterol representing the single most abundant compound. This observation is consistent with the structural complexity of the sperm plasma membrane. Metabolomic profiling similarly identified glycerophospholipids (44%), eicosanoids (14%) and N-acyl amino acids (12%) as the major metabolite classes. The integration of lipidomic and metabolomic data highlighted functionally interconnected pathways related to membrane dynamics, energy metabolism, and hormone biosynthesis. Overall, this work establishes a robust, sensitive, and scalable analytical framework that enables the high-coverage molecular characterization of spermatozoa from limited sample material, laying the groundwork for future biomarker discovery and clinical applications in male infertility research.
    Keywords:  MS/MS; lipidomics; male infertility; metabolomics; sperm
    DOI:  https://doi.org/10.3390/cells15070649
  6. Se Pu. 2026 Apr;44(4): 467-478
    [Determination of 38 antibiotic residues in honey by magnetic dispersive solid-phase extraction-liquid chromatography-tandem mass spectrometry].
    Jing Sun, Yu Zhang, Feng-Mao Liu, Wen Zhao, Yue Jin, Lin Zhang, Xiao-Feng Xue, Rui Chen, Hua-Tian Zhou.
      Accurate antibiotic residue detection in honey is crucial for food safety and veterinary drug regulation in apiculture. Honey is a particularly challenging matrix. The high sugar content， strong viscosity， and pronounced matrix effects of the sample make it difficult to extract and detect target analytes effectively. Moreover， multiple antibiotics are used in beekeeping， while existing residue standards often provide incomplete coverage. These limitations highlight the urgent need for a robust and environmentally friendly multi-residue analytical method. In this study， a green and sensitive method was developed for the simultaneous determination of 38 antibiotic residues， including chloramphenicol， quinolones， and sulfonamides， in honey. Sample preparation was based on magnetic dispersive solid-phase extraction （MDSPE） using a mixed-mode hydrophilic-lipophilic balance （mHLB） magnetic sorbent. This sorbent combines hydrophilic and hydrophobic interactions， enabling efficient extraction of chemically diverse antibiotics. Method optimization was performed systematically. A Plackett-Burman design was used to screen seven factors influencing extraction. The sorbent dosage， sample volume， and extraction time were identified as the most significant. These variables were further optimized by response surface methodology， which minimized the number of experimental runs while clarifying interactions among factors. The final protocol required only 10 mg of sorbent， 5 mL of sample extract， and 1 mL of methanol for elution. This miniaturized approach represents an environmentally sustainable method. Chromatographic separation was achieved on a Poroshell 120 SB-C18 column （100 mm×2.1 mm，2.7 μm） using a gradient of acidified methanol and water. Detection was carried out by high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） with electrospray ionization operated in both positive and negative modes， and multiple reaction monitoring ensured high sensitivity and selectivity. The method was fully validated. Excellent linearity was obtained for all analytes in the range of 0.02-200 μg/L， with correlation coefficients above 0.99. Limits of detection ranged from 0.01 to 0.12 μg/kg， while limits of quantification ranged from 0.02 to 0.39 μg/kg. Recoveries at three spiked levels ranged from 70.5% to 109.3%， with relative standard deviations （RSDs） below 10.8%. Matrix effects ranged from 0.82 to 1.19， indicating effective suppression of interference and reliable quantification in complex honey samples. The method was successfully applied to 42 commercial honey batches from different geographical regions. Several antibiotics were detected， including chloramphenicol， as well as other compounds without established maximum residue limits. These findings indicate potential misuse of antibiotics in beekeeping and reveal important gaps in current regulatory frameworks， underscoring the need for continuous monitoring and timely updates to residue standards. In addition to high sensitivity and wide applicability， the method demonstrated strong environmental advantages. Green performance was assessed using the AGREEprep tool， which evaluates twelve principles of sustainable sample preparation. The method achieved a high score of 0.73， reflecting minimal solvent consumption， low sorbent use， low energy demand， and negligible hazardous waste. These features align with the principles of green analytical chemistry and significantly reduce the environmental impact of residue analysis. In conclusion， this work establishes an MDSPE-HPLC-MS/MS for multi-class antibiotic residue detection in honey. The method combines simplicity， sensitivity， robustness， and environmental compatibility. It provides reliable technical support for risk assessment and regulatory improvement of antibiotic residues in honey， and it offers a valuable reference for the development of green analytical methods for trace contaminants in other complex food and environmental matrices.
    Keywords:  antibiotic residues; green analytical chemistry; honey; magnetic dispersive solid-phase extraction （MDSPE）; mixed-mode sorbent
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.07017
  7. Int J Mol Sci. 2026 Mar 26. pii: 3017. [Epub ahead of print]27(7):
    LC-MS/MS Method for Therapeutic Drug Monitoring of Abiraterone, Darolutamide, Apalutamide, Enzalutamide, and Metabolites in Prostate Cancer Patients.
    Bianca Posocco, Diletta Pasin, Nicoletta De Cesaro, Alice Pivetta, Sara Gagno, Giovanni Canil, Eleonora Cecchin, Riccardo Cecchin, Sara Speziani, Arianna Dri, Giorgia Bortolus, Michele Spina, Sandra Santarossa, Fabio Puglisi, Lucia Fratino, Erika Cecchin.
      Accurate measurement of androgen receptor pathway inhibitors (ARPIs) and their active metabolites is essential for pharmacokinetic studies and therapeutic drug monitoring (TDM) in patients with prostate cancer (PC). However, their simultaneous determination in human plasma is analytically challenging because of the wide concentration ranges. This study aimed to develop and validate a sensitive and robust LC-MS/MS method for the quantification of abiraterone, Δ4-abiraterone, enzalutamide, N-desmethyl enzalutamide, darolutamide, keto-darolutamide, apalutamide, and N-desmethyl apalutamide in human plasma. Sample preparation was performed by protein precipitation, followed by chromatographic separation and detection using multiple reaction monitoring with isotopically labeled internal standards (total run time 6.5 min). The method was validated in accordance with regulatory guidelines by assessing selectivity, linearity, sensitivity, accuracy, precision, recovery, matrix effects, and stability. The assay demonstrated good linearity (≥0.997) across clinically relevant concentration ranges, with lower limits of quantification between 0.1 and 40 ng/mL, depending on the analyte. Intra- and inter-day precision and accuracy were within acceptable limits, and recovery and matrix effects were consistent across different plasma matrices. Stability experiments conducted in plasma and whole blood provided practical guidance for sample handling. The method was successfully applied to 79 plasma samples from 61 patients with metastatic PC. Measured concentrations were generally consistent with published pharmacokinetic data, while unexpectedly high ABI levels were observed. Sample collection occurred between 1 and 28 h after the last drug intake, enabling assessment of the analytical method across the entire pharmacokinetic profile.
    Keywords:  ARPIs; analytical validation; mass spectrometry; prostate cancer; therapeutic drug monitoring
    DOI:  https://doi.org/10.3390/ijms27073017
  8. Analyst. 2026 Apr 15.
    Disentangling method-induced differences in metabolomics: single- versus dual-phase extraction.
    Sehyeon Erica Kim, Tao Huan.
      Efficient metabolite extraction is a key determinant of metabolome coverage and data quality in metabolomics. In this study, we systematically compare two widely used metabolite extraction strategies in untargeted metabolomics, including methanol-based single- and methyl tertiary-butyl ether (MTBE) based dual-phase solvent systems, and evaluate their performance across common urine and plasma, in liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. Through a rational experimental design, we disentangled the multifactorial differences between single- and dual-phase extractions into three analytically distinct components: pipetting, partitioning, and matrix effects. We further employ a comprehensive panel of isotopically labelled internal metabolite standards derived from 13C-labeled yeast extract to quantitatively characterize extraction-induced metabolic changes. Our results indicate that pipetting-induced variability is minimal, as polar standards exhibit fold changes close to unity between single- and dual-phase extraction protocols. In contrast, partitioning effects are strongly governed by solvent composition and phase volume ratios. For example, in plasma dual-phase extraction, where aqueous and organic phases are approximately equal, partitioning losses are limited. However, in urine dual-phase extraction, a higher organic-to-aqueous volume ratio promotes substantial redistribution of nonpolar metabolites into the organic phase. In addition, matrix effects further contribute to selective differences between the two methods, with ion suppression observed for subsets of compounds within specific retention time regions. Ultimately, the observed net response of each metabolite reflects the combined influence of partitioning behavior and matrix effects. Collectively, these findings highlight the complex, multifactorial nature of extraction-driven differences in LC-MS-based metabolomics. The framework established here can be readily extended to future method development, enabling systematic and mechanistic evaluation of method-induced metabolic changes.
    DOI:  https://doi.org/10.1039/d6an00025h
  9. STAR Protoc. 2026 Apr 10. pii: S2666-1667(26)00144-9. [Epub ahead of print]7(2): 104491
    Protocol for metabolite extraction and sample preparation from primary astrocytes for mass spectrometry-based metabolomic analysis.
    Ya-Gin Chang, Gong-Min Lin, Chih-Yu Lin, Yijuang Chern.
      Astrocytes play essential roles in supporting neuronal function, particularly through the regulation of brain energy metabolism. In response to physiological and pathological stimuli, astrocytes dynamically adjust their metabolic pathways and energy output. Here, we present a protocol for metabolite extraction and sample preparation from primary astrocytes for mass spectrometry analysis. We describe steps for integrating astrocyte culture and liquid chromatography-mass spectrometry (LC-MS) metabolite analysis to enable reproducible profiling of astrocytic energy metabolism under different experimental conditions. For complete details on the use and execution of this protocol, please refer to Chang et al.1.
    Keywords:  Cell culture; Metabolomics; Neuroscience; Protocols in Metabolomics and Lipidomics
    DOI:  https://doi.org/10.1016/j.xpro.2026.104491
  10. Anal Chim Acta. 2026 Jun 22. pii: S0003-2670(26)00379-X. [Epub ahead of print]1404 345429
    Rapid quantitative bioanalysis using LC-HRIM-QTOFMS with flexible sample delivery and variable traveling wave profiles.
    Sabrina M Cramer, Viktoria Kowarz, Diethard Mattanovich, Stephan Hann, Tim Causon.
       BACKGROUND: Incorporating an additional high-resolution ion mobility (HRIM) dimension can increase throughput and deliver results with high sensitivity and minimal ion suppression for bioanalytical LC-MS applications. However, the lengthy HRIM separation step conventionally comes at the cost of less data points per LC peak and a reduced working range, compromising quantification goals. This work addresses these challenges by using an alternative method for sample delivery and HRIM traveling wave profiles to establish methods for rapid quantification of small molecules and metabolites in complex samples.
    RESULTS: New HRIM-QTOFMS workflows for rapid, quantitative methods with sufficient sampling of LC peaks to harness high-resolution IM separation and obtain high quality analytical data were developed. Variable traveling wave profiles, which vary the applied amplitude and frequency over an HRIM scan, were used to increase the sampling of LC peaks to >15 points per peak. By optimizing LC sample introduction with feed injection, peak areas were improved and the total analysis times could be further reduced to <5 min per sample using short LC columns (3 to 5 cm). This allowed riboflavin produced by three different engineered strains of the yeast Komagataella phaffii to be quantified in supernatant and cell lysates in 5 min per sample, along with other flavin nucleotides. Additionally, phytosiderophores exuded by graminaceous plants (barley and sorghum) were identified and quantified in agreement with a validated LC-MS/MS method, and with an analysis time of 3 min per sample.
    SIGNIFICANCE: This study presents a new combination of rapid LC and HRIM separation dimensions with integrated feed injection mode for quantitative bioanalytical applications. We demonstrate that high-throughput methods for routine analysis of small molecules in biological samples can be realized by using HRIM traveling wave profiles to boost sampling across LC peaks and through post-HRIM fragmentation for the quantification of isomers.
    Keywords:  Biotechnology; High-throughput; Ion mobility; Phytosiderophores; Riboflavin; Structures for lossless ion manipulation
    DOI:  https://doi.org/10.1016/j.aca.2026.345429
  11. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 08. pii: S1570-0232(26)00140-6. [Epub ahead of print]1277 125051
    Optimisation and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for cannabis and its metabolites in urine.
    Min-Tz Weng, Qiuda Zheng, Angela Ratsch, Zeyang Zhao, Jared A Miles, Phong K Thai, Kathryn J Steadman.
      The two most abundant phytocannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Their major metabolites include 7-hydroxy-cannabidiol (CBD-OH), 7-carboxy-cannabidiol (CBD-COOH), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH) and 11-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). As THC and CBD metabolites are metabolised to their glucuronide conjugates, it is essential to account for these via enzyme de-conjunction during analysis of urine. The new generation of recombinant β-glucuronide enzymes have the potential to improve processing speed and hydrolysis efficiency for quantification of phytocannabinoids and their metabolites. This study aimed to optimise the de-glucuronidation and preparation of urine samples for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of phytocannabinoids and their metabolites, and use the method to quantify cannabinoids in authentic urine samples. Two β-glucuronidase enzymes (B-One and BGTurbo) were compared for their ability to hydrolyse the glucuronide forms of CBD-OH, CBD-COOH, THC-OH and THC-COOH. Enzyme volume was varied to optimise hydrolysis efficiency. Subsequent sample treatment using protein precipitation, filtration or a combination of both were compared. Hydrolysis efficiency was calculated, and validation parameters were measured. Treating a volume of 200 μL of urine with 200 μL of B-One enzyme, followed by protein precipitation using acetonitrile and filtration with a regenerated cellulose syringe filter, was optimal in terms of 100% hydrolysis efficiency and 93% cannabinoid recovery. The lower limit of quantification (LLOQ) was 0.2 ng/mL for CBD and THC, 1 ng/mL for CBD-OH and THC-OH, and 0.5 ng/mL for CBD-COOH and THC-COOH. Calibration curves extending to 100 ng/mL were linear with correlation coefficients exceeding 0.998; as the method involved a 6-fold dilution, this method can be applied to measurement of up to 600 ng/mL without additional dilution. Three authentic urine samples ranged in concentration of THC and CBD metabolites, from non-detectable up to a maximum of 385 ng/mL for THC-COOH. Single-step hydrolysis using B-One enzyme and combined precipitation with filtration for clean sample preparation allows for rapid cleavage of cannabinoid glucuronides with high recovery.
    Keywords:  CBD; Cannabinoids; Glucuronidase; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); THC
    DOI:  https://doi.org/10.1016/j.jchromb.2026.125051
  12. Se Pu. 2026 Apr;44(4): 403-412
    [Determination of ergot alkaloids toxins in forage grass by ultra performance liquid chromatography-tandem mass spectrometry coupled with solid-phase supported liquid-liquid extraction].
    Cai-Xia Ren, Xiao-Min Wang, Wan-Li Sheng, Yi-Zhi Han, Chun-Yan Zhang, Shu-Zhan Zheng.
      Toxic alkaloids， especially ergot alkaloid toxins， present in forage grass and pose serious health hazards to humans and livestock. Hence， a method for simultaneously detecting these dangerous plant toxins is needed. This study established a rapid method for detecting ten ergot alkaloid toxins using solid-phase supported liquid-liquid extraction technology combined with ultra performance liquid chromatography-tandem mass spectrometry. Forage samples were extracted with 8.0 mL of acetonitrile containing 1% formic acid. The extraction mixture was vortexed and then centrifuged at 8 000 r/min for 5 min， after which 1.0 mL of the supernatant was mixed with 3.0 mL of water and vortexed for 0.5 min. The entire mixture was transferred to a solid-phase supported liquid-liquid extraction column and allowed to stand for 10 min. The column was eluted with 6.0 mL of ethyl acetate and the collected eluate was evaporated under nitrogen at temperatures below 40 ℃. The residue was dissolved in 1 mL of acetonitrile-water （1∶3， volume ratio， containing 0.5% formic acid）， and separated using an Acquity UPLC BEH C18 column （100 mm×2.1 mm， 1.7 μm）. A solution of 0.5% formic acid in water containing 0.25 mmol/L ammonium acetate was used as mobile phase A， while 0.5% formic acid in acetonitrile was used as mobile phase B. The following gradient elution program was used： 0-1.0 min， 90%B； 1.0-5.0 min， 90%B-75%B； 5.0-10 min， 75%B-50%B； 10-12 min， 50%B-10%B； 12-14 min， 10%B； 14-16 min， 10%B-90%B. Ten ergot alkaloid toxins （including three pairs of isomers： ergocornine， ergocristine， ergocristinine， ergocryptine， and ergocryptinine） were effectively separated following gradient elution. Positive electrospray-ionization mode and multiple reaction monitoring （MRM） scanning was used for detection， with an external standard curve used for quantification purposes. The ten ergot alkaloid toxins exhibited good linear relationships within their respective linear ranges （r2>0.995）. Alfalfa， Leymus chinensis， oats， and silage corn exhibited LODs of 0.1-2.3 μg/kg for the ten ergot alkaloid toxins， with LOQs of 0.4-7.3 μg/kg. The developed method exhibited overall recovery rates of between 66.3% and 116.7%， with relative standard deviations of less than 9.9%. The matrix effect mainly manifested itself in the form of matrix suppression， with silage corn exhibiting a significantly stronger matrix effect than the other three types of forage. Silage corn exhibited a strong matrix effect in more than 50% of samples， while alfalfa， oats， and Leymus chinensis showed relatively low matrix effects， with more than 63% being weak. The developed method is simple to operate， provides accurate results， and exhibits minimal interference； hence， it is suitable for simultaneously screening and confirming ergot alkaloid toxins in forage， thereby providing technical support for monitoring forage quality， while also expanding the applicability of solid-phase supported liquid-liquid extraction technology in the toxin-detection field.
    Keywords:  ergot alkaloid toxins; forage grass; solid-phase supported liquid-liquid extraction; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.02014
  13. Biomed Chromatogr. 2026 Jun;40(6): e70425
    A High-Throughput LC-MS/MS Bioanalytical Method for Simultaneous Determination of Mirabegron and Solifenacin in Pharmacokinetic Investigations.
    Sadakar Tungapati, Srinivasu Nuvuluri, M Ramakrishna.
      Mirabegron, a Beta-3 adrenoceptor agonist drug, and Solifenacin succinate, an antimuscarinics drug, are used to treat overactive bladder in combination. Till today, no bioanalytical method has been reported to estimate Mirabegron and Solifenacin succinate in type of matrix. Mirabegron and Solifenacin succinate were measured in rat plasma using Darifenacin as the internal standard, a newly developed and validated bioanalytical LC-MS/MS technique. The analytes were separated using a Luna C18 column and a mobile phase combination of acetonitrile: 0.1% formic acid in HPLC water (50:50 v/v). The flow rate was maintained at an isocratic level of 1.0 mL/min, and the runtime was about 5 min. Mirabegron (m/z 397.51 to 83.78) and Solifenacin succinate (m/z 481.56 to 148.79) were determined by using the mass spectra with positive mode of multiple reaction monitoring. The strategy was approved in accordance with USFDA regulations. Accuracy with mean % recovery of 96.25% to 98.45% and 96.46% to 98.05% and strong linearity with r2 value of 0.9998 in the ranges of Mirabegron (2.5-100 ng/mL) and Solifenacin succinate (0.5-20 ng/mL) are the outcomes. All other parameters fall within the approved range. Indicators of medication effectiveness and safety, pharmacokinetic parameters, may be determined using the proposed technique.
    Keywords:  LC–MS/MS; Mirabegron; Solifenacin succinate; USFDA guidelines; rat plasma
    DOI:  https://doi.org/10.1002/bmc.70425
  14. J Sep Sci. 2026 Apr;49(4): e70409
    A Sensitive and Selective UHPLC-MS/MS Method Based on Mercaptopropionic Acid Derivatization for Quantification of Juglone in Biological Matrices.
    Xinyue Zheng, Yue Deng, Si Cheng, Di Lu, Yibo Wang, Zichang Tian, Hongyu Xue, Lei Yin, Meiyun Shi.
      In this study, a highly sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of juglone, a poorly ionizable naphthoquinone, in biological matrices. To enhance ionization efficiency, pre-column derivatization was employed using sulfhydryl nucleophiles (β-mercaptoethanol and 3-mercaptopropionic acid) via Michael addition. Derivatization with mercaptopropionic acid significantly improved mass spectrometric response (50-fold increase) and minimized matrix effects compared to mercaptoethanol. The method, validated per FDA guidelines, exhibited excellent linearity (r ≥ 0.995) from 3 to 150 ng/mL, with accuracy within -10.6% to 4.3% and precision between 1.29% and 5.34%. The lower limit of quantification was 3 ng/mL. Extraction recovery and matrix effects ranged from 99.84% to 102.06% and 92.75% to 98.98%, respectively, with derivatives demonstrating good stability. Cellular uptake studies in MCF-7 cells revealed time-dependent but limited intracellular accumulation of juglone, with the majority retained in the culture medium. This robust derivatization-based UHPLC-MS/MS approach provides a reliable tool for investigating juglone's pharmacokinetics and toxicological mechanisms.
    Keywords:  cytotoxicity; juglone; liquid chromatography–tandem mass spectrometry; pharmacokinetic study; pre‐column derivatization
    DOI:  https://doi.org/10.1002/jssc.70409
  15. Se Pu. 2026 Apr;44(4): 479-485
    [Rapid determination of lubabegron residues in animal-derived foods by ultra performance liquid chromatography- tandem mass spectrometry].
    Cheng Yang, Wei-Xia Zhu, Ya-Feng Liu, Hong-Wei Zhang, Wei Wei, Kai Hu.
      Lubaberone （LUB） is the first U S Food and Drug Administration （FDA） approved feed additives for reducing gaseous emissions from animals or their wastes， and the intake of animal-derived foods is an important source of human exposure to LUB. However， the lack of applicable analytical methods makes it difficult for regulatory authorities to monitor LUB in animal-derived foods. There is an urgent need to establish efficient and accurate methods for the analysis of LUB in animal-derived foods. In this study， a method was developed for the determination of LUB from six types of typical animal-derived foods （beef， bovine liver， bovine fat， mutton， sheep liver and sheep fat） by ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. The effects of extraction solvent types， extraction methods， extraction time and purification cartridges on the recovery of LUB were investigated， and the optimal conditions for sample pretreatment were confirmed. The homogenized samples were extracted using 0.5% formic acid acetonitrile via ultrasonication for 10 min， and then filtered by high-speed centrifugation， and the extracted solution was cleaned using SPE with a PRiME HLB cartridge. The LUB was separated on a Shim-pack GIST C18-AQ chromatographic column （100 mm×2.1 mm， 2.7 μm） with 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile as mobile phases at a flow rate of 0.3 mL/min， and detected in positive ion switching mode （ESI+） with multiple reaction monitoring （MRM） scanning， and quantitatively analyzed using the external standard method. Under the optimized experimental conditions， LUB in different animal-derived food matrices showed good linearity within their respective linear concentration ranges with the correlation coefficients （r） greater than 0.99. The limits of detection （LODs） and the limits of quantification （LOQs） were 0.4‒1.0 μg/kg and 1.0‒2.0 μg/kg， respectively. The recoveries of LUB spiked at low， medium and high levels ranged from 81.5% to 116.5% with relative standard deviations （RSDs） of 2.0%‒8.2%. The method is simple， rapid and highly sensitive， which can enable the analysis of LUB residues in animal-derived foods， and provides analytical technology support for the daily detection of LUB residues in imported and exported animal-derived foods.
    Keywords:  beef; lubabegron （LUB）; solid-phase extraction （SPE）; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.02006
  16. Anal Chim Acta. 2026 Jun 22. pii: S0003-2670(26)00385-5. [Epub ahead of print]1404 345435
    Development of analytical methods for the quantification and enhanced detection of unsaturated short-chain FAHFAs.
    Rachana M Gangadhara, Perumalsamy Parasuraman, Divyavani Gowda, Hariom Yadav, Ganesh V Halade, Shu-Ping Hui, Siddabasave Gowda B Gowda.
      Short-chain fatty acid esters of hydroxy fatty acids (SFAHFAs) comprise a novel class of endogenous lipids predominantly localized in the gut and play essential roles in metabolic and inflammatory regulation. Previous studies have enabled chemical synthesis and quantitative analysis of saturated SFAHFAs using validated analytical methods. However, the detection and quantification of unsaturated SFAHFAs remains challenging because of their synthetic complexity and inherently low abundance in biological matrices. In this study, a comprehensive in-house library comprising thirty isomeric unsaturated SFAHFAs was synthesized to facilitate the development of two LC-MS/MS based analytical approaches: (i) direct analysis without derivatization for reliable quantification and (ii) a sensitivity-enhanced method based on 2-(2-pyridyl) ethylamine derivatization. Liquid chromatography-tandem mass spectrometry was optimized for sensitive and selective quantification, demonstrating excellent linearity with limits of detection between 0.5 and 5 fmol and limits of quantification between 5 and 50 fmol. The non-derivatized method was used to profile fecal SFAHFAs in murine and human samples. In mice, exercise increased the levels of both saturated and unsaturated SFAHFAs, whereas an obesogenic diet selectively reduced saturated SFAHFA levels. By contrast, analysis of fecal samples from humans with mild cognitive impairment revealed no significant differences from those of healthy controls, suggesting potential disease- and matrix-specific variability. A chemical derivatization approach using 2-(2-pyridyl) ethylamine enhanced detection sensitivity by approximately 1000-fold with LOD between 0.0005 and 0.001 fmol and LOQ 0.005 and 0.01 fmol. This study presents an analytical approach for the synthesis, quantification, and improved detection of unsaturated SFAHFAs, establishing a foundation for the further exploration of their roles in metabolic disorders associated with gut dysbiosis.
    Keywords:  Chemical synthesis; Liquid chromatography; Mass spectrometry; Obesogenic diet; PEA derivatization; SFAHFA
    DOI:  https://doi.org/10.1016/j.aca.2026.345435
  17. J Chromatogr A. 2026 Apr 09. pii: S0021-9673(26)00317-1. [Epub ahead of print]1777 466987
    A chromatographic approach to avoid the matrix effect caused by sodium acetate clusters in the liquid chromatography coupled with electrospray ionization mass spectrometry analysis of free fatty acids in human plasma.
    Yuki Fukushima, Kazuhiro Yamamoto, Koichi Machida, Akira Kotani, Hideki Hakamata.
      Liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) is widely used for the analysis of biological samples in pharmacokinetic studies, clinical diagnostics, and therapeutic drug monitoring. However, matrix effects caused by coexisting substances in samples often compromise analytical sensitivity and accuracy. Our study showed that sodium acetate clusters, formed from sodium ions in human plasma and acetate ions in mobile phase, interfered with the detection of free fatty acids (FFAs) in a reversed-phase (RP) LC-ESI-MS analysis. To address this issue, we employed a trimodal column (reversed-phase/weak anion-exchange/weak cation-exchange), which effectively separates the sodium ions from FFAs and successfully reduces the matrix effects. Owing to its high compatibility with conventional octadecylsilyl (ODS) bonded column, enabling easy implementation into existing ODS-based methods, the trimodal column offers a promising new chromatographic strategy for mitigating matrix effect caused by sodium acetate clusters in RPLC-ESI-MS bioanalysis.
    Keywords:  Bioanalysis; Free fatty acid; Matrix effect; RPLC-ESI-MS; Sodium acetate cluster
    DOI:  https://doi.org/10.1016/j.chroma.2026.466987
  18. J Pharm Biomed Anal. 2026 Apr 01. pii: S0731-7085(26)00156-1. [Epub ahead of print]277 117488
    GC-MS method development and validation for the determination of Short Chain Fatty Acids in human feces.
    Gkanali Vasiliki, Farantatou Konstantina, Begou Olga, Theodoridis Georgios, Gika Helen, Virgiliou Chistina.
      Short Chain Fatty Acids (SCFAs), the end products of microbial fermentation of dietary fibers, appear to be key mediators of the beneficial effects elicited by the gut microbiome and have been shown to exert multiple effects on metabolism. In this study, we developed and validated a sensitive, accurate, and reproducible GC-MS method for the simultaneous quantification of SCFAs (Acetic acid (C2), propionic acid (C3), butyric acid (C4), isobutyric acid and isovaleric acid) in human feces. Sample preparation was simplified while maintaining robustness, following systematic evaluation of homogenization, extraction solvents, and acidification conditions. The optimized method demonstrated high analytical performance, with limits of detection ranging from 0.01 to 0.52 μmol/g and good precision and accuracy in accordance with FDA and EMA bioanalytical guidelines Stability studies revealed that SCFAs remain stable in acidified fecal samples for up to 10 days without cold-chain requirements, while -80 °C storage was optimal for long-term preservation and 4 °C suitable for short-term handling. The applicability of the method was confirmed through analysis of samples collected from healthy volunteers. Overall, the developed approach provides a practical, high-throughput, and scalable tool for SCFA analysis, supporting applications in clinical research, metabolomics, and large-scale microbiome studies.
    Keywords:  Biomarkers; Gut-brain axis; Host-guest co-metabolism; Metabolic profiling; SCFAs; Symbiosis
    DOI:  https://doi.org/10.1016/j.jpba.2026.117488
  19. Anal Bioanal Chem. 2026 Apr 14.
    Establishment and application of a QuEChERS-based high-performance liquid chromatography-mass spectrometry method for the analysis of 56 per- and polyfluoroalkyl substances in human plasma.
    Yujing Chuai, Xiaotao Zhou, Yuting Zhang, Jing Zhang, Jiaqi Guo, Yu Wang, Qianru Zhou, Yu Xia, Chunying Luo, Li Yong, Xiaoli Zou.
      Per- and polyfluoroalkyl substances (PFAS) have attracted increasing concern due to their environmental persistence and potential adverse health effects. In this study, a high-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of 56 PFASs in human plasma. After the plasma sample was extracted with methanol, 5 mg primary secondary amine (PSA), 20 mg graphitized carbon black (GCB), and 30 mg C18 were added for QuEChERS purification, followed by nitrogen drying and reconstitution. The chromatographic separation was performed on a C18 column with a gradient elution and was quantified on mass spectrometry with electrospray ionization in negative mode. The method was validated and good linearity was obtained in the range of 0.050-50.0 ng/mL. The limits of detection were 0.0024 to 2.05 ng/mL, and except for a few perfluoroalkyl phosphinic acids and fluorinated polymer-based substances with low signal intensities, most PFASs showed satisfactory recoveries of 70-120%, with relative standard deviations below 12%. The validated method was applied to analyze plasma samples from pregnant women and males over 50 years old. The results revealed widespread PFAS contamination in the population, with perfluoroalkyl carboxylic acids as the dominant contributors, followed by perfluoroalkyl sulfonic acids and accompanied by a limited number of emerging PFAS alternatives. There are population-specific differences in PFAS exposure, with median concentrations typically higher in males aged over 50 years than in pregnant women, but 6:2 FTS is much higher in pregnant women. The established method is sensitive and efficient, and the results highlight the importance of population-stratified biomonitoring of PFASs.
    Keywords:  HPLC–MS/MS; PFAS exposure biomonitoring; Plasma analysis; QuEChERS
    DOI:  https://doi.org/10.1007/s00216-026-06486-2
  20. Foods. 2026 Apr 03. pii: 1220. [Epub ahead of print]15(7):
    Determination of Glycidol in Soy Sauce Using p-Dimethylaminophenol Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry.
    Yifan Zhao, Peng Wang, Longlong Wang, Lixia Qin, Hai Chi, Guangxin Yang, Xiaosheng Shen, Chengqi Fan, Xiaoqing Tian, Mian Hasnain Nawaz, Cong Kong.
      Glycidol, a probable human carcinogen, remains an under-investigated process contaminant in soy sauce. This study developed a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for its determination in this complex condiment. The approach combined chemical derivatization with p-Dimethylaminophenol hydrochloride for analyte stabilization with an optimized sample pretreatment using a custom-packed activated carbon solid-phase extraction (SPE) cartridge effectively removed matrix interferences, and performing the derivatization at pH 6.5 prevented conversion of 2- and 3-monochloropropanediol (2-MCPD and 3-MCPD) into glycidol, ensuring high specificity and accuracy. This approach shows broad linearity from 1 to at least 100 ng/mL (R2 = 0.9993), and demonstrates excellent performance, with a limit of detection and quantification of 0.5 ng/mL and 1.0 ng/mL, respectively. Application to commercial samples (n = 11) confirmed the presence of glycidol, highlighting the need for its monitoring. This work provides a robust analytical tool essential for supporting food safety surveillance of this contaminant in fermented foods.
    Keywords:  HPLC-MS/MS; carbon yarn; derivatization; food contaminant; glycidol; p-Dimethylaminophenol; soy sauce
    DOI:  https://doi.org/10.3390/foods15071220
  21. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 05. pii: S1570-0232(26)00138-8. [Epub ahead of print]1277 125049
    Development and validation of an LC-MS/MS assay for serum 5α-androstane-3α, 17β-diol 17-glucuronide with enhanced interference resolution.
    André Filipe Rodrigues-Oliveira, Andrea Tedesco Faccio, Gustavo Arantes Rosa Maciel, Karina Helena Morais Cardozo, Valdemir Melechco Carvalho.
      5α-androstane-3α,17β-diol 17-glucuronide (3α-diol G) is a testosterone metabolite and an important biomarker of hormonal dysregulation, including polycystic ovary syndrome (PCOS) and virilizing syndromes in women such as idiopathic hirsutism. While immunoassay kits are commonly used for its quantification in clinical laboratories, these methods are prone to interference due to the structural similarity among steroidal compounds. In this study, we developed and validated a robust LC-MS/MS method for the quantification of 3α-diol G in human serum. Extraction was performed using a μelution SPE approach, allowing the achievement of a suitable LLOQ (0.140 ng/mL) with a low sample volume (100 μL). The chromatographic method successfully separated major known interferences, including glucuronidated androsterone (ADT-G) and etiocholanolone (Etio-G), and resolved unknown coeluting compounds that could otherwise lead to overestimation. Additionally, monitoring the transitions of the most common phospholipid showed no interference from these species. Method validation followed CLSI recommendations, assessing imprecision, accuracy, linearity, limit of quantification, carryover, and stability. The routine analysis incorporated a system suitability protocol based on ion ratio monitoring to detect potential coeluting interferences prior to batch analysis. This work highlights the importance of chromatographic separation and ion ratio monitoring in steroid analysis and provides a reliable alternative to immunoassay-based quantification of 3α-diol G.
    Keywords:  Bioanalytical method validation; Interference resolution; Ion ratios; Selectivity; Steroidal hormones; System suitability
    DOI:  https://doi.org/10.1016/j.jchromb.2026.125049
  22. J Pharm Biomed Anal. 2026 Mar 30. pii: S0731-7085(26)00155-X. [Epub ahead of print]277 117487
    Heterologous internal calibration for multiplexed internal quantification in targeted LC-MS/MS bioanalysis.
    Oriane Strassel, Max Feinberg, Gioele Visconti, Julien Boccard, Serge Rudaz.
      Absolute quantification is crucial in bioanalysis for both clinical and non-clinical applications but remains challenging, especially for endogenous metabolites. In external calibration, the absence of a true blank matrix complicates calibration curve preparation. Multi-targeted internal calibration (IC) using stable isotope-labeled standards (SILs) as one-point calibrants offers an attractive alternative, yet its use is limited by the availability and cost of homologous SILs. To address this, heterologous SILs can serve as surrogate calibrants for multiple analytes, relying on response factors (RFs) established between each analyte and selected SILs in the matrix of interest. This study assessed the suitability of such an alternative isotopic-dilution strategy, referred to as heterologous internal calibration (H-IC), within an LC-MS/MS method developed for chronic kidney disease (CKD) research. A panel of 18 metabolites and their corresponding SILs was initially analyzed to generate reference values, and the performance of various analyte-SIL combinations was compared with that of the original homologous internal calibration procedure. Validation indicated satisfactory accuracy and precision for most analytes, with trueness generally within 70-130% and precision below 10%. Only a few low-abundance metabolites showed minor deviations. Finally, reducing the calibration panel to five heterologous SILs for quantifying all 18 analytes in 30 patient samples produced results comparable to those obtained using individual homologous SILs. These findings demonstrate that H-IC can substantially reduce SIL requirements and analytical costs while maintaining acceptable quantitative performance for metabolite measurement in human plasma.
    Keywords:  Absolute quantification; Endogenous metabolites; Heterologous internal calibration; LC–MS/MS; Response factor; Stable isotope-labeled standards
    DOI:  https://doi.org/10.1016/j.jpba.2026.117487
  23. Front Mol Biosci. 2026 ;13 1765747
    Pitfalls of onco-metabolomics: impact of sample integrity on metabolomic investigations in more than 4500 human serum samples from ten different cohorts.
    Michael Ladurner, Selina Strathmeyer, Tobias Ameismeier, Helmut Klocker, Eberhard Steiner, Gerhard Aigner, Martin Puhr, Tina Böld, Diana Drettwan, Franziska Sommermeyer, Iris E Eder.
       Introduction: Metabolomics such as nuclear magnetic resonance spectroscopy or mass spectrometry (MS) in different body fluids are considered potentially useful diagnostic techniques for various diseases including cancer. One of the most important prerequisites of metabolomics is a high sample quality, for which reason explicit care must be taken during pre-analytical/analytical handling.
    Methods: In the present study, we investigated the influence of pre-processing (PPT), and pre-centrifugation time (PCT), sample storage time (SST), and sample texture on NMR-based metabolite levels in 4,658 long-term and short-term stored retrospectively and prospectively collected serum samples from breast and prostate cancer patients as well as from healthy men.
    Results: We found that the majority of the metabolites were highly stable with regard to variations in PCT, PPT, or SST. PCT and PPT significantly affected the concentrations of only a few individual metabolites, including ascorbic acid, asparagine, glucose, glutamic acid, glutamine, lactate, phenylalanine, pyruvic acid, and serine, indicating that pre-analytical protocol variations need to be considered for the quantitative analysis of metabolites. Notably, the glucose:lactate and glutamine:glutamic acid ratios were found to be suitable to assess sample quality in case of high PCT or PPT. Importantly, the highest sample quality was detected in prospectively collected serum samples with strict protocol adherence and a total PPT of only 1.2 h. Specific care must also be taken with the analysis of lipemic samples, in which strong variations in the concentrations of lipid metabolites, albumin, and valine were observed.
    Discussion: In summary, our data show that the majority of metabolites are mostly stable with regard to variations in pre-analytical processing, indicating that retrospective biobank samples are suitable for metabolomics studies. However, individual metabolites are strongly dependent on PCT and PPT, suggesting that a short PPT may be mandatory for clinical diagnosis, depending on the individual metabolite to be measured.
    Keywords:  NMR spectroscopy; biobank; breast cancer; human serum; metabolomics; pre-analytical/analytical variations; prostate cancer; sample integrity
    DOI:  https://doi.org/10.3389/fmolb.2026.1765747
  24. Anal Chim Acta. 2026 Jun 22. pii: S0003-2670(26)00395-8. [Epub ahead of print]1404 345445
    Therapeutic oligonucleotide (nusinersen) metabolism in cerebrospinal fluid samples of patients with spinal muscular atrophy based on liquid chromatography coupled with mass spectrometry data.
    Sylwia Studzińska, Anna Lemska, Jakub Szymarek, Maria Mazurkiewicz-Bełdzińska.
       BACKGROUND: Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder caused by a deficiency of the survival motor neuron protein. The introduction of antisense oligonucleotide therapy has markedly improved prognosis, particularly following approval of Nusinersen (Spinraza), the first SMA drug. Although modified with 2'-O-methoxyethyl and phosphorothioate groups, nusinersen is metabolized. Comprehensive characterization of these metabolites in cerebrospinal fluid is limited due to analytical challenges associated with antisense oligonucleotides. This study aimed to develop a first liquid-liquid/solid-phase extraction procedure combined with ion-pair ultra-high-performance liquid chromatography coupled with mass spectrometry for the extraction, separation, and identification of nusinersen and its metabolites in cerebrospinal fluid samples from SMA patients treated with Spinraza.
    RESULTS: A two-step sample preparation procedure provided high nusinersen recovery (89.2 ± 1.8%), repeatability, eliminated matrix effects, and enabled 50-fold sample concentration. All of these proved essential for the detection and identification of low-abundance metabolites collected four months after dosing. Reliable metabolite identification requires sufficient chromatographic resolution, especially for metabolites differing by a single nucleotide with similar nominal m/z values. Consequently, careful ion-pair reagent selection and MS optimization are crucial for improving separation and sensitivity to detect low-abundance metabolites. Our results showed that propylamine and dimethylbutylamine may be used interchangeably, but the second one provides higher resolution. Accurate mass measurement and characteristic fragment ions derived from methylated nucleobases and phosphorothioate groups ensured reliable identification. Analysis of CSF samples from 17 pediatric SMA (at various dosing stages) patients revealed extensive in vivo metabolism, predominantly via 3'-exonucleolytic cleavage, yielding multiple N-shortmers. For the first time, interpatient variability was observed in nusinersen detectability and metabolite profiles of CSF samples.
    SIGNIFICANCE: To our knowledge, this is one of the first comprehensive studies demonstrating the applicability of the developed procedure to observe differences in the metabolism of nusinersen and, in the future, linking them to therapeutic effects, therapeutic monitoring, or the patient's condition. The methodology addresses key bioanalytical challenges (reproducibility, purification, concentration, separation) and enables metabolite profiling in a complex biological matrix. Moreover, it provides a foundation for future investigations linking metabolism, drug exposure, and clinical response in SMA therapy.
    Keywords:  Cerebrospinal fluid; Extraction; Ion pair ultra high liquid chromatography coupled with mass spectrometry; Metabolites; Nusinersen; Spinal muscular atrophy; Therapeutic effect
    DOI:  https://doi.org/10.1016/j.aca.2026.345445
  25. Anal Bioanal Chem. 2026 Apr 14.
    Development of electromembrane extraction using deep eutectic solvents for 22 representative veterinary drug residues in honey, milk, and eggs.
    Junling He, Shaoyun Peng, Shangjun Xie, Yong Tang, Chiliang Lin, Stig Pedersen-Bjergaard, Frederik André Hansen, Jing Zhang, Chen Zhou, Haimin Zou.
      The widespread occurrence of veterinary drug residues in animal-derived foods demands analytical methods that are not only sensitive and reliable, but also efficient and environmentally sustainable. To meet this need, this study developed a novel high-throughput platform based on 96-well electromembrane extraction (EME) using a designed deep eutectic solvent (DES) composed of 6-methylcoumarin and thymol (1:1, w/w) as a green and efficient supported liquid membrane. When coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), the proposed DES-EME method enabled the simultaneous determination of 22 veterinary drugs in three kinds of complex food samples: honey, milk, and eggs. Under optimized conditions (10 mM trifluoroacetic acid, 50 V, 15 min), the method demonstrated excellent analytical performance, featuring wide linear ranges of 0.01-100 ng/mL with r ≥ 0.993, and limits of quantification (LOQs) expressed as original sample concentrations of 1.0 ng/g for honey and 25 ng/g for milk and eggs. Recoveries ranged from 40 to 99%, with relative standard deviations (RSDs) below 13% for most analytes. An AGREEprep score of 0.67 confirmed the greenness of the method, highlighting strengths in energy efficiency, sample throughput, and sustainability. Collectively, this work demonstrates that DES-EME represented a viable and sustainable approach for monitoring veterinary drug residues in complex food matrices.
    Keywords:  Deep eutectic solvents; Electromembrane extraction; Food analysis; Sample preparation; Veterinary drug residues
    DOI:  https://doi.org/10.1007/s00216-026-06497-z
  26. bioRxiv. 2026 Apr 08. pii: 2026.04.06.716604. [Epub ahead of print]
    Comparison of Extraction Methods for the Quantification of Phytohormones from Tomato Fruits and Leaves by LC-MS/MS.
    Carlos A Juarez Guzman, Linxing Yao, Corey D Broeckling, Cristiana T Argueso.
      Accurate, simultaneous, and efficient quantification of chemically diverse phytohormone species is a critical task towards understanding the complex system of phytohormone signaling pathways. Quantification of phytohormones with the commonly used technique liquid chromatography coupled to tandem mass spectrometry is susceptible to the influence of non-phytohormone components present in the sample, a phenomenon referred to as matrix effect. To reduce matrix effect, some phytohormone quantification methods include additional steps of cleanup of crude extracts. However, to what extent additional purification steps provide increased accuracy compared to simpler, less laborious methods is seldomly evaluated. We evaluated three previously described phytohormone extraction methods, two of which include solid-phase extraction and one that does not, in their ability to minimize matrix effect and generate accurate estimates of phytohormone species spanning six classifications, from fruit and leaf tissue of Solanum lycopersicum cv. Micro-Tom (tomato). Our results show that, while the methods that included solid phase extraction occasionally outperformed each other regarding matrix effect and/or recovery efficiency for broad range of phytohormones, they rarely outperformed the simpler single-phase extraction method.
    Short Abstract: Accurate, simultaneous quantification of chemically diverse phytohormones by LC-MS/MS is frequently confounded by matrix effects, leading to the incorporation of additional purification steps. We systematically compared three published extraction protocols with or without solid-phase extraction in tomato tissues across six hormone classes. Solid-phase methods occasionally improved matrix suppression or recovery, but did not consistently outperform the single-phase approach, questioning the added value of extra cleanup steps, particularly when high-throughput is desired, as in the case of systems biology interrogations.
    DOI:  https://doi.org/10.64898/2026.04.06.716604
  27. MAK Collect Occup Health Saf. 2025 ;10(4): Doc070
    Tri-(2-ethylhexyl) trimellitate - Determination of 1-MEHTM, 2-MEHTM, 5OH-1-MEHTM, 5OH-2-MEHTM, 5cx-1-MEPTM, and 5cx-2-MEPTM in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
    MAK Commission
      The working group "Analyses in Biological Materials" of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) developed and verified this biomonitoring method for the measurement of six specific metabolites of the plasticiser tri‑(2‑ethylhexyl) trimellitate (TEHTM) in urine. Specifically, this method determines two monoester isomers as primary hydrolysis products of TEHTM, 1‑mono-(2‑ethylhexyl) trimellitate (1‑MEHTM) and 2‑mono-(2‑ethylhexyl) trimellitate (2‑MEHTM), as well as the oxidatively formed secondary derivatives, namely 1‑mono-(2‑ethyl-5‑hydroxyhexyl) trimellitate (5OH‑1‑MEHTM), 2‑mono-(2‑ethyl-5‑hydroxyhexyl) trimellitate (5OH‑2‑MEHTM), 1‑mono-(2‑ethyl-5‑carboxypentyl) trimellitate (5cx‑1‑MEPTM), and 2‑mono-(2‑ethyl-5‑carboxypentyl) trimellitate (5cx‑2‑MEPTM). Determination is carried out after enzymatic hydrolysis of the urine sample as well as enrichment of the analytes by online SPE. Via integrated, automatic column-switching, the analytes are transferred onto the analytical column in backflush mode, separated by liquid chromatography, and quantified by tandem mass spectrometry. Calibration is performed using calibration standards prepared in pooled urine and processed analogously to the samples to be analysed. The following isotope-labelled substances are added to the urine samples as internal standards: D5‑1‑MEHTM, D5‑2‑MEHTM, D5‑5OH‑1‑MEHTM, D5‑5cx‑1‑MEPTM, and D5‑5cx‑2‑MEPTM. The method provides reliable and accurate analytical results, as shown by the good precision data with standard deviations no greater than 8%. Good accuracy data were obtained with mean relative recoveries in the range of 97-109%. The method is both selective and sensitive, and provides quantitation limits in the range of 0.04-0.12 μg/l.
    Keywords:  LC-MS/MS; TEHTM; biomonitoring; urine
    DOI:  https://doi.org/10.34865/bi331931e10_4or
  28. Se Pu. 2026 Apr;44(4): 413-421
    [Determination of eight ultraviolet absorbers in surface water and wastewater by ultra performance liquid chromatography-triple quadrupole mass spectrometry].
    Hui-Jing Sun, Bei-Bei Zhang, Meng-Qiao Huang, Hui Wang, Guan-Jiu Hu, Hui-Min Chen.
      Ultraviolet （UV） absorbers are a group of chemicals widely used in various industrial and consumer products， such as plastics， coatings， and personal care products， to protect against UV radiation. Among them， benzotriazole derivatives （e.g. UV-326， UV-327， UV-328， UV-329， and UV-P） are the most frequently employed. Owing to their widespread use and potential persistence in the environment， these compounds have been detected in various environmental matrices， including surface water， wastewater， sediment， and biota. Certain UV stabilizers have been reported to exhibit endocrine-disrupting properties and pose potential ecological risks. Therefore， developing sensitive and reliable analytical methods for monitoring these compounds in environmental samples is essential. To address the need for reliable detection methods， this study developed a robust method based on liquid-liquid extraction （LLE） coupled with ultra performance liquid chromatography-triple quadrupole mass spectrometry （UPLC-MS/MS） for the simultaneous determination of eight UV absorbers in surface water and wastewater. Critical optimization of the pretreatment process focused on solvent selection and purification parameters. The finalized protocol involved extracting 100 mL water samples twice with dichloromethane. After nitrogen-assisted solvent evaporation， the residue was reconstituted in methanol and mixed with the internal standard solution. UPLC-MS/MS parameters were optimized to achieve optimal instrumental performance. The separation of the eight UVs was performed using a BEH C18 column （100 mm × 2.1 mm， 1.7 μm） with a gradient elution system consisting of 0.2% （mass fraction） formic acid aqueous solution and acetonitrile at a flow rate of 0.4 mL/min. The injection volume was 2 μL. Detection was performed in positive ion mode using multiple reaction monitoring （MRM）， with an electrospray ionization voltage set at 5 500 V. Quantification was achieved via internal standard calibration to ensure precision and accuracy. The method demonstrated excellent linearity for all target compounds across their respective concentration ranges， with a correlation coefficient （r）>0.995. The method detection limits （MDLs） ranged from 1.3 ng/L to 2.8 ng/L， indicating high sensitivity. Recovery tests conducted at low， medium， and high spiking levels （20， 200， and 800 ng/L） yielded recoveries of 80.3%-117.8%， with relative standard deviations （RSDs） of 1.4%-10.5%， confirming the method's robustness across different sample matrices. Application of the method to 10 textile dyeing wastewater samples revealed the presence of four UV absorbers： UV-329， UV-326， UV-328， and UV-350. Notably， UV-329 showed the highest detection frequency and accounted for 85% of the total detected mass concentrations， ranging from 5.2 to 2 109 ng/L. Its prevalence suggests its widespread use in industrial processes and potential persistence in aquatic environments. In conclusion， the developed method is highly sensitive， accurate， and reliable for detecting UV absorbers in environmental water samples. Its successful application to surface water and wastewater analysis provides a valuable tool for monitoring these emerging contaminants， thereby supporting the assessment of their environmental and health risks. This study highlights the importance of continued monitoring and regulation of UV absorbers to mitigate their potential adverse effects on ecosystems and human health.
    Keywords:  liquid-liquid extraction （LLE）; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）; ultraviolet （UV） absorbers
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.08001
  29. MAK Collect Occup Health Saf. 2025 ;10(2): Doc046
    Mycotoxins - Determination of deoxynivalenol and deepoxydeoxynivalenol in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
    MAK Commission
      The working group "Analyses in Biological Materials" of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) developed and verified the presented biomonitoring method. The aim of this method is the selective and sensitive quantitation of deoxynivalenol (DON; free DON plus glucuronides not otherwise specified) and its metabolite deepoxydeoxynivalenol (DOM‑1) in urine. After enzymatic hydrolysis of the urine sample and purification of the analytes on an immunoaffinity column, followed by preconcentration of the eluates under a stream of nitrogen, determination is carried out by high-performance liquid chromatography-tandem mass spectrometry (LC‑MS/MS). Calibration is performed with comparative standards prepared in urine and treated analogously to the samples to be analysed. DON is quantified using an internal standard (ISTD; 13C15‑DON), whereas DOM‑1 is quantified without the use of an ISTD. Good precision data with standard deviations below 9% for DON and below 6% for DOM‑1, as well as good accuracy data with mean relative recoveries in the range of 93-114% for DON and 97-103% for DOM‑1, show that the method provides reliable and accurate analytical results. The method is both selective and sensitive, and has a limit of quantitation of 0.179 μg/l for DON and of 0.26 μg/l for DOM‑1. Due to rapid renal excretion, the method is primarily suitable for analysing acute exposure which occurred only hours prior to sampling.
    Keywords:  LC-MS/MS; biomonitoring; deoxynivalenol; mycotoxins; urine
    DOI:  https://doi.org/10.34865/bi5148110e10_2or
  30. Nat Biotechnol. 2026 Apr 15.
    Structure-centric searching enables global mapping of the public metabolome.
    Yasin El Abiead, Jeong In Seo, Vincent Charron-Lamoureux, Michael Strobel, Wilhan Donizete Gonçalves Nunes, Haoqi Nina Zhao, Kine Eide Kvitne, Simone Zuffa, Helena Mannochio-Russo, Harsha Gouda, Cristina Bez, Abubaker Patan, Shipei Xing, Jasmine Zemlin, Ipsita Mohany, Julius Agongo, Andres Mauricio Caraballo Rodriguez, Lindsey A Burnett, Victoria Deleray, Abzer K Pakkir Shah, Jarmo-Charles Kalinski, Daniel Petras, Nikiforos Alygizakis, Jeremy Carver, Ozgur Yurekten, Thomas Payne, Eoin Fahy, Shankar Subramaniam, Juan Antonio Vizcaíno, Mingxun Wang, Pieter C Dorrestein.
      Searching and learning from aggregated public metabolomics data spanning thousands of studies remained largely inaccessible. Here we present StructureMASST, a web-based application enabling scalable, structure-centric searches across public metabolomics repositories using molecule names or chemical representations. It queries a precomputed knowledgebase of 2.19 billion spectral matches and 420 million metadata links, supports modification-tolerant and mass-shift searches, and maps chemical structures across taxonomy, biological context and environmental conditions to accelerate discovery.
    DOI:  https://doi.org/10.1038/s41587-026-03082-8
  31. Anal Chem. 2026 Apr 13.
    Rapid Cold Fixation of Mouse Colon (Col'RFix) Enables High-Resolution Mass Spectrometry Imaging.
    Sabina H Skov, Henrik M Jensen, Ole N Jensen.
      Cellular-scale MALDI mass spectrometry imaging (MSI) requires tissue preparation strategies that preserve both tissue morphology and native molecular integrity. Mouse colon poses a particular challenge as fresh-frozen sections are mechanically fragile and frequently lose architectural definition, whereas formalin-fixed paraffin-embedded-based approaches require retrieval steps that can compromise molecular signals. Here, we present Col'RFix (Colon Rapid Fixation), a rapid, liquid-based fixation workflow optimized for transverse mouse colon tissue. Col'RFix employs short-duration cold fixation (4% paraformaldehyde at 5 °C for 10 min) combined with low-melting agarose embedding to provide mechanical support during cryosectioning. Protocol validation assessed morphology preservation, analyte diffusion, molecular coverage, and spatial localization using replicate colon sections from two high-fat diet mice analyzed across multiple MSI runs at 5, 10, and 20 μm pixel sizes, three MALDI-MSI modalities, and in positive and negative ion modes. Feasibility of biological comparison was evaluated in an independent cohort of lean (n = 3) and high-fat diet (n = 3) mice. Col'RFix enabled reproducible imaging down to 5 μm pixel size, supporting spatial lipidomics of fatty acids and multiple lipid classes with minimal delocalization. Compared to unfixed tissue sections sampled within 1 cm of the same region, Col'RFix preserved fine colon morphology, as confirmed by H&E staining, allowing precise correlation of molecular features with histological details. Spectral comparison of fixed and unfixed tissues indicated largely conserved molecular profiles, with compound-dependent signal attenuation. Together, these results establish Col'RFix as a reproducible workflow, enabling compartment-resolved molecular maps of mouse colon at 5 μm pixel size.
    DOI:  https://doi.org/10.1021/acs.analchem.5c04968
  32. Anal Chem. 2026 Apr 17.
    Decoding the Oxylipin Chemical Space Using Ion Identity Molecular Networking.
    Sandra M Camunas-Alberca, Francesco Bartolini, Hana Cermakova, Ángel Sánchez-Illana, David Perez-Guaita, Pablo Cañadas-Miquel, Alberto Gil-de-la-Fuente, Jean-Marie Galano, Thierry Durand, Ana Gradillas, Coral Barbas.
      Oxylipins are bioactive lipid mediators that play key roles in biological and pathological processes. Their remarkable chemical diversity makes their identification by untargeted LC-MS/MS analyses challenging. To date, effective solutions for their comprehensive characterization remain unavailable. Here, we present the first implementation of the recently refined Ion Identity Molecular Networking (IIMN) strategy to map the chemical space of oxylipins, together with a systematic evaluation of factors that hinder accurate annotation in MS/MS datasets. Building on recent mzmine software developments, we implemented a fully local strategy to perform the IIMN analysis without requiring web platforms or external tools. We established a high-quality MS/MS spectral library from 67 commercially available oxylipin standards using LC-MS data obtained in data-dependent acquisition mode. Integrating the detailed characterization of ion species generated during electrospray ionization into IIMN reduced network complexity. Across configurations, the modified cosine algorithm proved most effective for separating full-length from cyclized forms and for clustering oxylipins through structurally coherent relationships. Application of the IIMN workflow to mouse spleen extracts, in combination with our in-house and publicly available experimental MS/MS libraries, enabled the organization of oxylipins into molecular families, facilitating their structural characterization and the discovery of novel species. Although manual curation remained necessary for certain coeluting isomers and ambiguous fragments, the IIMN-based approach significantly improved network interpretability and understanding. Overall, this study establishes IIMN as a robust bioinformatic tool for decoding oxylipin diversity and provides a successful strategy for mapping their chemical space, characterizing them within samples, and discovering novel mediators in biological matrices. The new combined reference spectral library has been made publicly available and will serve as a valuable resource for future redox lipidomics research.
    DOI:  https://doi.org/10.1021/acs.analchem.5c06084
  33. J Chromatogr B Analyt Technol Biomed Life Sci. 2026 Apr 10. pii: S1570-0232(26)00163-7. [Epub ahead of print]1277 125074
    Ultrafiltration-MSPE with in-situ quaternary aminooxy oximation for LC-MS/MS quantification of nine serum free androgens.
    Xianhua Zhang, Lingling Liu, Huiyu Xu, Xin Xiong, Rongsheng Zhao, Libo Zhao, Rong Li, Li Yang.
      Accurate quantification of circulating androgens, particularly the biologically active free fraction, is analytically challenging due to ultra-low concentrations and extensive protein binding in human serum. Here, we developed and validated an integrated ultrafiltration-LC-MS/MS workflow for simultaneous quantification of nine endogenous serum free androgens. Free fractions were isolated by phosphate-buffered saline dilution followed by temperature-controlled centrifugal ultrafiltration (37 °C) using pretreated regenerated-cellulose membranes to minimize non-specific adsorption. Ultrafiltrates were purified and enriched by magnetic solid-phase extraction (MSPE), and in-situ oximation derivatization with a quaternary aminooxy reagent was applied to enhance electrospray response. The validation data demonstrated pg/mL-level sensitivity with LLOQ of 0.5-10 pg/mL, good linearity (r > 0.99), acceptable matrix effects, and high recovery. The method was applied to serum samples from 86 female patients (21-50 years) undergoing infertility evaluation, showing higher free testosterone, 11β-hydroxyandrostenedione, and androstenedione in an AMH-enriched PCOS-suspicion subgroup versus a control group. Overall, this ultrafiltration-MSPE-in-situ derivatization LC-MS/MS method provides a practical and robust platform for multiplexed measurement of serum free androgens at pg/mL levels for clinical research applications.
    Keywords:  In-situ oximation derivatization; LC–MS/MS; Magnetic solid-phase extraction (MSPE); Polycystic ovary syndrome (PCOS); Serum free androgens; Ultrafiltration
    DOI:  https://doi.org/10.1016/j.jchromb.2026.125074
  34. MAK Collect Occup Health Saf. 2025 ;10(2): Doc045
    Mycotoxins - Determination of aflatoxins, ochratoxin A, free ochratoxin α, gliotoxin, citrinin, and dihydrocitrinone in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
    MAK Commission
      The working group "Analyses in Biological Materials" of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) developed and verified the presented biomonitoring method. The aim of this method is the selective and sensitive quantitation of aflatoxins (aflatoxins B1, B2, G1, G2, M1), ochratoxin A (OTA), free ochratoxin α (OTα), gliotoxin (GT), citrinin (CIT) and dihydrocitrinone (DH‑CIT) in urine. Sample preparation comprises enrichment and purification of the analytes by solid-phase extraction using OASIS HLB cartridges. Calibration is performed with comparative standards prepared in pooled urine and treated analogously to the samples to be analysed. The aflatoxins, OTA, CIT, and DH‑CIT are quantified using isotope-labelled internal standards (ISTDs), whereas OTα and GT are quantified without an ISTD. Determination is carried out by high-performance liquid chromatography-tandem mass spectrometry (LC‑MS/MS). The method provides reliable and accurate analytical results, as shown by the good precision data with standard deviations below 9% for the aflatoxins, OTα, GT and CIT, below 13% for OTA, and below 20% for DH‑CIT. Good accuracy data were obtained with mean relative recoveries in the range of 93-107% for the aflatoxins, OTα, GT and CIT, in the range of 83-103% for OTA, and in the range of 81-108% for DH‑CIT. The method is both selective and sensitive, and has quantitation limits in the range of 0.013-0.022 μg/l for the aflatoxins and OTA and a quantitation limit of 1.0 μg/l for OTα, 1.5 μg/l for GT, 0.0075 μg/l for CIT, and 0.01 μg/l for DH‑CIT.
    Keywords:  LC-MS/MS; aflatoxins; biomonitoring; citrinin; gliotoxin; mould toxin; mycotoxins; ochratoxin A; urine
    DOI:  https://doi.org/10.34865/bi116265e10_2or
  35. J Endocr Soc. 2026 May;10(5): bvag076
    Determining EDC exposure: direct analytical methods are essential for accurate analysis of human biospecimens.
    Roy Gerona, Alia Sovereign, Deborah French, Mikayla Joyce Gonzaga, Frederick S Vom Saal, Patricia Hunt.
       Purpose: Estimates of human exposure are a major component of chemical risk assessment. Studies of bisphenol A (BPA) have raised concern that exposure has been underestimated because the lack of standards for the measurement of the major BPA metabolites has necessitated the use of flawed analytical tools to indirectly estimate them. Because other endocrine-disrupting chemicals (EDCs) are measured using similar indirect methods, we evaluated the accuracy of indirect analysis for representatives from three different classes of non-persistent EDCs that undergo rapid phase II metabolism: bisphenols, parabens, and phthalates.
    Methods: A direct LC-MS/MS method that simultaneously measures bisphenol S (BPS), propyl paraben (PrP), and monobutyl phthalate (MBP), and their major metabolites in urine, was used to quantify these EDCs in sixty second-trimester human urine samples. The same samples were also analyzed with a widely used indirect method that requires enzymatic hydrolysis before estimating metabolite levels.
    Results: Marked discrepancies were evident when maternal urine samples were analyzed by both direct and indirect methods. Indirect analysis underestimated levels of all three EDCs and BPA, with the magnitude of underestimation varying by analyte.
    Conclusion: The accuracy of widely used "indirect" analytical methods that estimate metabolite levels in human urine is neither predictable nor consistent. Greater precision and accuracy is attained using authentic standards for metabolites. Given the importance of biomonitoring data in estimating human EDC exposure, analytical accuracy is critical. Availability of standards for both the parent compound and its major metabolites should be required before a chemical enters the marketplace.
    Keywords:  analytical chemistry; biomonitoring; endocrine disrupting chemicals (EDCs); exposure; risk assessment
    DOI:  https://doi.org/10.1210/jendso/bvag076
  36. MAK Collect Occup Health Saf. 2025 ;10(1): Doc021
    Glyphosate - Determination of glyphosate and AMPA in urine by LC-MS/MS: Biomonitoring Method - Translation of the German version from 2025.
    MAK Commission
      The working group "Analyses in Biological Materials" of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area developed and verified the presented biomonitoring method. The aim of this method is the selective and sensitive quantitation of glyphosate (N-phosphonomethylglycine) and its only metabolite, aminomethylphosphonic acid (AMPA), in urine. Samples undergo solid-phase extraction prior to liquid chromatography-tandem mass spectrometry using glyphosate-2-13C,15N and AMPA-13C,15N,D2 as internal standards. Calibration is carried out with urine from persons with no known exposure to glyphosate and AMPA. The procedure has been comprehensively validated and the reliability data have been confirmed by replication and verification of the procedure in a second, independent laboratory. Good precision data with standard deviations of 1.3-9.8% for glyphosate and 1.9-5.4% for AMPA, as well as good accuracy data with mean relative recoveries in the range of 91-102% for glyphosate and 100-106% for AMPA, show that the method provides reliable and accurate analytical results. The method is both selective and sensitive, and the limits of quantitation of 0.1 μg/l for glyphosate and 0.5 μg/l for AMPA are sufficient to determine occupational exposure as well as some of the background exposure in the general population.
    Keywords:  AMPA; LC-MS/MS; biomonitoring; glyphosate; urine
    DOI:  https://doi.org/10.34865/bi107183e10_1or
  37. Anal Sci Adv. 2026 Jun;7 e70083
    Wooden-Tip Electrospray Ionization Mass Spectrometry Combined With Machine Learning for Differentiating Thyroid Tumours.
    Da-Sheng Liu, Li Liu, Baixue Wang, Jianfeng Zhang, Hong-Guo Lin, Xiang-Xiong Huang, Kang-Jian Deng, Yu-Teng Zhou, Yunlong Pan, Bin Hu, Xue-Yang Huang.
      Direct mass spectrometry (MS) analysis of human tissues at the molecular level has great potential for clinical diagnosis and biomarker discovery. However, conventional MS-based analytical methods often require complicated and time-consuming sample preparation, which limits their applicability in rapid clinical analysis. In this study, we developed a rapid analytical strategy by integrating ambient ionization MS with machine learning (ML) for the differentiation of different thyroid tumours. A disposable slim wooden tip (WT) was employed as both a sample holder and an electrospray emitter, enabling direct extraction and ionization of metabolites from tiny thyroid tissue samples under electrospray ionization (ESI) conditions. Using this WT-ESI-MS method, lipid profiles of thyroid tissues could be obtained within minutes without extensive sample preparation. A total of 45 thyroid samples, including 15 healthy tissues, 15 benign tumours and 15 malignant tumours, were analysed. The acquired MS data were further processed using ML-based classification models to distinguish different tumours and identify potential lipid biomarkers. Structural characterization of representative lipids was also performed by MS/MS analysis. The results demonstrated that this WT-ESI-MS combined with ML provides a rapid and effective approach not only for differentiating tumour tissues and healthy samples but also for benign and malignant tumours, highlighting its potential application in clinical diagnosis and intraoperative tissue evaluation.
    Keywords:  electrospray ionization; machine learning; mass spectrometry; thyroid cancer; tissue analysis
    DOI:  https://doi.org/10.1002/ansa.70083
  38. Environ Pollut. 2026 Apr 09. pii: S0269-7491(26)00436-7. [Epub ahead of print]398 128066
    Uncovering tire wear signatures in urban aerosols: application of ultrahigh-performance liquid chromatography-tandem mass spectrometry using multimode ionization source for the determination of tire wear compounds.
    Mahmoud M Yassine, Ewa Dabek-Zlotorzynska, Alana Greaves.
      Tire wear compounds (TWCs) are an important source of pollution in urban environments, generated through the abrasion of vehicle tires and contributing significantly to airborne non-exhaust emissions. The major risk for human health is primarily related to the inhalation of TWCs emitted into the atmosphere which can trigger a wide range of adverse health effects. In this study, a rapid and comprehensive analytical method was developed using ultrahigh pressure liquid chromatography-triple quadrupole mass spectrometry via atmospheric pressure electrospray-chemical multi-mode ionization source (UPLC-ESCi-MS/MS) for the simultaneous quantitation of four benzotriazoles (BTRs), seven benzothiazoles (BTHs), three p-phenylenediamines (PPDs) and 1,3-diphenyl guanidine (DPG) in under 8 min. The ESCi source provided enhanced sensitivity for all target analytes relative to conventional electrospray ionization (ESI). Sample preparation was simple and utilized isotopically labelled internal and surrogate standards. All target TWCs exhibited high stability in MeOH during extraction. The method was demonstrated to be robust for the analysis of particulate matter with aerodynamic diameters less than 30 μm (PM30), between 10 and 2.5 μm (PM10-2.5) and less than 2.5 μm (PM2.5) collected at a traffic-influenced site. DPG was the most abundant TWC across all size fractions. Among BTRs, only benzotriazole (BTR) and 5-methyl benzotriazole (5-MeBTR) were detected in all fractions, while 2-hydroxy benzothiazole (2-HOBTH) was the dominant BTH. For all fractions, the ΣPPDs made up the smallest proportion of TWC. Overall, clear differences in the distribution of TWCs were observed in PM size fractions collected in a dense traffic area. The potential applications of this method for monitoring of TWCs will provide new traffic-related source data for environmental and public health studies. In addition, it will support the development and implementation of effective policies and mitigation measures to control the release of TWCs into the atmosphere, thereby reducing their harmful effects on people living in the vicinity of roadways.
    Keywords:  BTRs and BTHs; DPG; PPDs; TWCs; UPLC-ESCi-MS/MS; Urban atmospheric PM
    DOI:  https://doi.org/10.1016/j.envpol.2026.128066
  39. J Pharm Biomed Anal. 2026 Apr 12. pii: S0731-7085(26)00179-2. [Epub ahead of print]277 117511
    Validation of a quantitative analysis method for tryptophan metabolites in asthmatic mice and bacteria using GC-MS/MS.
    Weichen Xu, Mengqing Shang, Zichen Luo, Xinyu Ji, Yiwen Shan, Qingqing Chen, Jinjun Shan, Zhicheng Zhang.
      This study established a gas chromatography-tandem mass spectrometry (GC-MS/MS) method for the simultaneous quantification of 25 tryptophan (TRP) metabolites encompassing the kynurenine pathway, serotonin pathway, and indole pathway. The method was systematically validated in terms of precision and accuracy, demonstrating robust reliability. Application of this method to bacterial cultures as well as serum and lung tissues from asthmatic mice revealed significant alterations in TRP metabolic profiles. Owing to its high throughput and sensitivity, this method is well suited for the analysis of TRP metabolites in complex biological matrices and provides a robust platform for comprehensive profiling of TRP metabolites in complex biological matrices, facilitating studies of TRP metabolism in asthma.
    Keywords:  Asthma; GC-MS/MS; Tryptophan metabolism
    DOI:  https://doi.org/10.1016/j.jpba.2026.117511
  40. Anal Chem. 2026 Apr 17.
    Isotope Dilution NanoLC-MS/MS Quantitation of Methylglyoxal DNA-Protein Cross-Links: Formation and Repair in Human Cells.
    Reinner O Omondi, Elijah M Barnes, Krishna C Gurajala, Gabrielle Fisette, Ibaad A Chaudray, Luke Erber.
      DNA-protein cross-links (DPCs) represent a prevalent form of DNA damage that forms when cellular proteins become covalently trapped to DNA strands upon exposure to various endogenous and exogenous agents. Methylglyoxal is an endogenous metabolite that reacts with guanine and adenine bases in DNA and RNA, as well as cysteine, arginine, and lysine residues in proteins, generating advanced glycation end-products (AGEs), including DPCs. These modifications have been linked to human disease, including cancer, liver disease, diabetes, and neurodegenerative disorders. Herein, we present a mass spectrometry method for quantifying MGO-induced DNA-protein cross-links (DPCs) in human cells. We prepared an isotope 15N213C6-dG-MGO-Lys internal standard and developed a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting and quantifying the formation and repair of dG-MGO-Lys DPCs in cells. Genomic DNA was extracted, subjected to sequential protease and nuclease digestion, purified by offline high-performance liquid chromatography (HPLC), and analyzed by LC-MS/MS. The method's standard curve showed a strong linear relationship across a concentration range of 10-1000 fmol (R2 = 0.9994). The method achieved limits of detection (LOD) and quantification (LOQ) of 10 and 20 fmol, respectively. Inhibition of proteasome and SPRTN activity revealed that SPRTN functions as a predominant proteolytic enzyme in MGO DPC repair. Overall, this analytical approach can offer valuable insights into the relevance of DPCs in diseases linked to elevated MGO levels.
    DOI:  https://doi.org/10.1021/acs.analchem.5c06852
  41. J Chromatogr A. 2026 Apr 08. pii: S0021-9673(26)00312-2. [Epub ahead of print]1777 466982
    Comprehensive profiling of alkaloids in Buxus sinica by integrating global natural products social molecular networking with derivatization-assisted characteristic ion filtering.
    Sen Li, Song Wang, Zhongzhe Cheng.
      Comprehensive characterization of alkaloids in Buxus sinica (B. sinica) is hindered by extensive structural diversity, high isomeric complexity, and generally poor ionization efficiency in mass spectrometry. In this study, an integrated LC-MS/MS strategy combining global natural products social (GNPS) molecular networking of non-derivatized extracts with a derivatization-assisted characteristic ion filtering (CIF) workflow was established to achieve the most comprehensive alkaloid profiling of B. sinica reported to date. GNPS molecular networking enabled efficient dereplication and visualization of structurally related alkaloids based on MS/MS spectral similarity, facilitating network-guided annotation of major constituents. In parallel, chemical derivatization with 4-(N, N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole introduced predictable diagnostic fragment ions, markedly enhancing ionization efficiency and fragmentation specificity. A Python-based CIF algorithm was further developed to automate data preprocessing and selectively extract spectra containing characteristic ions, substantially improving annotation efficiency and coverage. By integrating these complementary approaches, a total of 56 alkaloids were characterized in B. sinica, including 21 compounds identified by GNPS analysis and 44 compounds revealed through derivatization-CIF, with 9 compounds detected by both workflows. This coverage exceeds previously reported studies on B. sinica alkaloids and highlights the complementary strengths of molecular networking and derivatization-driven data filtering. Overall, this work provides a robust and scalable analytical platform for exhaustive alkaloid profiling in complex botanical matrices and offers a valuable reference for natural product research and pharmaceutical analysis.
    Keywords:  Alkaloids; Buxus sinica; Characteristic ion filtering; Chemical derivatization; GNPS molecular networking
    DOI:  https://doi.org/10.1016/j.chroma.2026.466982
  42. Rapid Commun Mass Spectrom. 2026 Jul 15. 40(13): e70079
    A New Potential Biomarker for Strychnine Misuse in Human Urine Using Quadrupole-Orbitrap LC-MS/MS.
    Jianghai Lu, Jiarui Wang, Xuxiao Zhao, Qiaoling Fei, Xiaopei Wu, Jing Jing, Yang Liu.
      In this study, we investigate the metabolic profile of strychnine in human urine following controlled administration using liquid chromatography-quadrupole-Orbitrap mass spectrometry. A total of 25 metabolites were characterized and identified. These included 21 previously unreported and 4 previously reported metabolites. Four unreported metabolic pathways were discovered, namely, reduction, methylation, glycosylation, and glucuronidation. Among these, hydroxylation was identified as the major metabolic pathway. The detection windows in the urine for all 25 metabolites were compared with that of the parent drug. Metabolite S10 (2,3-dimethoxy-strychnine) was proposed as a novel potential biomarker for strychnine misuse, rather than strychnine itself, due to its longer detection time and higher number of strychnine-positive time points (exceeding 50 ng/mL in human urine) compared to the parent compound after oral administration. The identification of S10 extends the detection window and offers critical insights for doping control applications.
    Keywords:  doping control; metabolic profile; quadrupole Orbitrap LC–MS/MS; strychnine
    DOI:  https://doi.org/10.1002/rcm.70079
  43. Talanta. 2026 Mar 26. pii: S0039-9140(26)00381-4. [Epub ahead of print]308 129725
    Collision energy consistency between analyte and internal standard: A critical factor for accurate quantification of steroid hormones by LC-MS/MS.
    Xiaoli Ma, Wei Luo, Yixuan Cai, Ming Li, Qian Cheng, Songlin Yu, Ling Qiu.
      Simultaneous quantification of steroid hormones with vastly differing physiological concentrations using LC-MS/MS often necessitating compromised collision energy (CE) settings, potentially introducing inaccuracy. This study investigates the critical impact of CE consistency between analytes and their stable isotope-labeled internal standards (SIL-IS) on quantification accuracy using a triple quadrupole LC-MS/MS system. We analyzed six hormones in 61 plasma samples using a novel dual-CE acquisition method, monitoring SIL-IS response at both low and optimal CE. The method was reapplied to 70 patient samples following collision cell replacement to assess instrument dependency. Significant instrument-specific bias (15-92%) were observed for high-concentration analytes when CE settings between native analytes and SIL-IS were mismatched (LCE-HCE). These biases were markedly amplified on a degraded instrument. Synchronizing CE settings (LCE-LCE) or utilizing consistently optimized isotopic transitions restored accuracy, reducing mean bias to below 15% post-optimization. The instrument in optimal condition showed minimal CE-dependent bias. Inconsistent CE application is a hidden source of quantitative inaccuracy in multi-analyte steroid panels, exacerbated by suboptimal instrument conditions. While these findings provide important methodological insights, further studies on different LC-MS/MS platforms are warranted to establish their generalizability. We strongly recommend verification of CE consistency during method validation and instrument troubleshooting.
    Keywords:  Collision energy consistency; Internal standard; Isotope dilution mass spectrometry; LC-MS/MS method validation; Steroid hormone quantification
    DOI:  https://doi.org/10.1016/j.talanta.2026.129725
  44. Molecules. 2026 Mar 26. pii: 1090. [Epub ahead of print]31(7):
    Simultaneous LC-MS Profiling of Bioactive Ecdysteroids in Nutrient-Dense Plant Sources and Dietary Supplements.
    Velislava Todorova, Stanislava Ivanova, Raina Ardasheva, Kalin Ivanov.
      Phytoecdysteroids have garnered increasing interest due to their broad biological and pharmacological properties. The present study reports on the development and validation of a reliable liquid chromatography-mass spectrometry method for the detection and quantification of 20-hydroxyecdysone, turkesterone, and ponasterone. The optimized procedure improved ionization efficiency and chromatographic resolution through gradient elution using 0.1% formic acid in water and acetonitrile. Data acquisition in selective ion monitoring modes ensured high analytical precision, reproducibility, and sensitivity. The method demonstrated excellent linearity, accuracy, repeatability, and low detection limits, making it suitable for routine phytochemical and quality control applications. Application of the method to extracts from nutrient-rich superfoods, including kaniwa, spinach, quinoa, and asparagus, confirmed these plants as natural sources of phytoecdysteroids. Additionally, thirteen commercially available dietary supplements labeled as containing extracts of Rhaponticum carthamoides, Cyanotis arachnoidea, Ajuga turkestanica, or ecdysteroids were analyzed. Several products standardized to 80-95% ecdysterone contained substantially lower amounts than declared, with measured 20-hydroxyecdysone levels ranging from below the limit of detection to approximately 50 mg per capsule, whereas some non-standardized products exhibited moderate to high levels, reaching up to approximately 105 mg per capsule. Variability in turkesterone content was also observed among products marketed as standardized extracts. The method provides a simple, reliable, and accessible approach for the quantitative analysis of major phytoecdysteroids in complex plant matrices and dietary supplements. Its implementation may support phytochemical research, routine quality control, and anti-doping monitoring of ecdysteroid-containing products.
    Keywords:  LC–MS; dietary supplements; ecdysterone; phytoecdysteroids; quality control; sport; superfoods; turkesterone; validation
    DOI:  https://doi.org/10.3390/molecules31071090
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