bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–11–09
35 papers selected by
Sofia Costa, Matterworks
  1. Absolute quantification of polar metabolites using matrix-free calibration curves and post-column infusion of standards in HILIC-MS based metabolomics.
  2. An UPLC-MS/MS method for the simultaneous quantification of 6 steroids from the androgen "backdoor pathway" in human plasma.
  3. [Determination of 51 per- and polyfluoroalkyl substances in raw water and drinking water by online solid-phase extraction coupled with ultra performance liquid chromatography- tandem mass spectrometry].
  4. Impact of Preprocessing and data analysis strategies on metabolite annotation in biological samples: A mass spectrometry-based metabolomics study using feature-based molecular networking.
  5. Development and Clinical Validation of a Simultaneous Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry Assay for Three Triazole Antifungal Agents and Two Metabolites Enabling Precision Therapeutic Drug Monitoring in Chinese Patients With Hematologic Malignancies.
  6. Validation of a highly sensitive assay for the determination of rivastigmine in human plasma for pharmacokinetic studies.
  7. An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of mycophenolic acid glucuronide in human serum and plasma.
  8. An LC-MS/MS method for simultaneous quantification of pyrotinib, docetaxel and paclitaxel in human plasma and its application to therapeutic drug monitoring.
  9. An isotope dilution-liquid-chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of total and free valproic acid in human serum and plasma.
  10. Comparative evaluation of comprehensive offline 2D-LC strategies coupled to MS for untargeted metabolomic studies of human urine.
  11. A Fragmentation behavior-guided UHPLC-Q-Orbitrap HRMS method for the quantitative analysis of 26 perfluoroalkyl substances and their alternatives in water.
  12. Method development and validation for quantitative determination of urinary biomarkers of food intake for multiple foods.
  13. Development and validation of an LC-MS/MS method for Tirzepatide, a dual GIP/GLP-1 receptor agonist, in rat plasma for application to a pharmacokinetic study.
  14. [Development of Enantioselective Imaging Method for Chiral Molecules by on-Tissue Chiral Derivatization and Ion Mobility Mass Spectrometry].
  15. [Quantitation of cow's milk allergen in hypoallergenic infant formulas using ultra-high performance liquid chromatography- tandem mass spectrometry].
  16. Automated dual-cartridge solid-phase extraction method for multi-residue determination of veterinary drugs in bovine muscle, liver, fat, and milk.
  17. A large language model for deriving spectral embeddings for accurate compound identification in mass spectrometry.
  18. Integrating mass spectrometry and hyperspectral imaging for protoporphyrin IX detection in malignant glioma tissue.
  19. [Determination of eight per- and polyfluoroalkyl substances in seawater using dispersive membrane extraction-ultra-high performance liquid chromatography-tandem mass spectrometry].
  20. Establishment of a LC-MS/MS method for the simultaneous quantitative determination of trimethylamine N-oxide and four precursors in human saliva.
  21. Not One Method to Rule Them All: A Comparative Study of Chromatographic Platforms (RP-LC-, HILIC-, SFC-, and IC-HRMS) for Water Analysis.
  22. Molecules to medicine: advances in metabolomics for natural product drug discovery.
  23. Exploring ammonium salt doping to enhance ion abundance for quantitative mass spectrometry imaging.
  24. [Determination of genotoxic impurity in the bulk drug of crizotinib by high performance liquid chromatography-electrospray ionization tandem mass spectrometry].
  25. Full-Signal Ultrahigh-Resolution NMR by Parameter Estimation.
  26. A bio-SPME-LC-MS/MS method for the quantitative determination of cannabinoids in plasma samples: analytical strategy for optimization of matrix modification.
  27. Pharmaceutical Metabolite Data Base, PharmMet DB: Reference data base for drug metabolites generated by human liver S9 fraction.
  28. [Magnetic one-step purification-high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry for the determination of 54 veterinary drug residues in carp].
  29. Direct Identification and Quantification of Flavonoids and Their Structural Isomers Using Ambient Ionization Tandem Mass Spectrometry.
  30. High-resolution dual-polarity molecular imaging platform unraveling spatial metabolic heterogeneity in multiple plant tissues.
  31. Targeted polar metabolomics data for Neurospora crassa wild type and mutant strains cultured on glucose.
  32. [Development of a Qualitative Analytical Method for Milk, Egg, Buckwheat, and Peanut Allergens Using LC-MS/MS].
  33. At-line analysis of antisense oligonucleotide purified fractions using fast chromatographic methods.
  34. Forced Degradation Studies of Dobutamine and Isolation, Identification, and Characterization of Potential Degradation Impurity by Preparative HPLC, LC-MS/MS. Development and Validation of Stability Indicative UPLC Method.
  35. Novel LC-MS/MS strategies combining low-temperature partition extraction and DLLME for sensitive levamisole determination in fish.
  1. J Chromatogr A. 2025 Oct 31. pii: S0021-9673(25)00851-9. [Epub ahead of print]1764 466507
    Absolute quantification of polar metabolites using matrix-free calibration curves and post-column infusion of standards in HILIC-MS based metabolomics.
    Mengle Zhu, Lieke Lamont, Pascal Maas, Amy C Harms, Marian Beekman, P Eline Slagboom, Anne-Charlotte Dubbelman, Thomas Hankemeier.
      Polar metabolites play essential roles in inflammation, immune regulation, and metabolism, and have emerged as important biomarkers in clinical research. However, accurate absolute quantification of these metabolites in complex biological matrices using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) remains challenging, due to matrix effects that compromise measurement accuracy. While (stable isotope-labeled-) internal standards ((SIL-)IS) are commonly used to correct these effects, their limited availability and high cost can hinder their applicability. In this study, we present a robust approach using HILIC-MS coupled with post-column infusion of standards (PCIS) to enable absolute quantification of polar metabolites in plasma, using calibration curves prepared in neat solution without using SIL standards. First, the optimal PCIS for each metabolite was identified based on absolute matrix effect (AME) values, and method performance was systematically evaluated in terms of linearity, precision, accuracy, and matrix effects. Results showed that the PCIS approach consistently achieved comparable results to (SIL-)IS correction for analytes with SIL standards, and superior performance for analytes without available SIL standards. Application to a study cohort demonstrated consistency between this HILIC-PCIS-MS method and Nuclear Magnetic Resonance (NMR) quantification. Validation using NIST SRM 1950 plasma confirmed high quantification accuracy (80 % - 120 %) for most metabolites using a matrix-free calibration curve combined with PCIS in HILIC-MS. In summary, PCIS offers a promising and cost-effective alternative to (SIL-)IS correction, particularly benefiting metabolites lacking SIL standards, and provides a strategy for accurately correcting matrix effects and advancing quantitative metabolomics in complex biological matrices.
    Keywords:  Absolute quantification; HILIC-MS; Matrix effects; Polar metabolites; Post-column infusion of standards
    DOI:  https://doi.org/10.1016/j.chroma.2025.466507
  2. Clin Chim Acta. 2025 Nov 05. pii: S0009-8981(25)00579-0. [Epub ahead of print] 120700
    An UPLC-MS/MS method for the simultaneous quantification of 6 steroids from the androgen "backdoor pathway" in human plasma.
    Yongqi Liu, Susu Cai, Tingting Zhang, Xujing Kong, Jianfeng Zhou, Baorong Chen, Zenghe Li.
       BACKGROUND: Hyperandrogenism poses significant diagnostic challenges in women's healthcare, requiring precise identification of elevated androgen sources and types for accurate diagnosis and optimal treatment. Key steroid metabolites of the androgen backdoor pathway, which are critical for differentiating androgen sources, remain underutilized due to constraints in measurement methodologies. We developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantifying six backdoor pathway steroids in human plasma.
    METHODS: Plasma samples (500 μL) underwent liquid-liquid extraction with ethyl acetate/hexane (8:2, v/v). Steroids were separated on a C18 column using methanol-water gradient elution with 0.05 % (v/v) formic acid and analyzed by positive electrospray ionization in multiple reaction monitoring (MRM) mode. Total runtime was 6.2 min. The method was validated following the CLSI C62-A guidelines.
    RESULTS: Lower limits of quantification for six steroids ranged from 0.0138 to 0.0538 nmol/L. Linearity was excellent (R2 ≥ 0.996). Intra- and inter-day coefficients of variation were < 8 %, and recoveries ranged from 85.8 % to 110.5 %. Preliminary application to healthy controls and patients with congenital adrenal hyperplasia (CAH), including hyperandrogenic cases [21-hydroxylase deficiency (21-OHD) and 11β-hydroxylase deficiency (11β-OHD)] and hypoandrogenic cases [17α-hydroxylase deficiency (17α-OHD)], demonstrated the method's utility in identifying the type and source of androgen dysregulation.
    CONCLUSION: This sensitive, rapid method enables simultaneous quantification of six key steroids in the androgen backdoor pathway, thereby providing enhanced metabolic profiling for patients with hyperandrogenism and improving diagnostic capabilities for androgen disorders.
    Keywords:  Androgen; Backdoor pathway; Hyperandrogenism; Liquid chromatography-tandem mass spectrometry; Steroids
    DOI:  https://doi.org/10.1016/j.cca.2025.120700
  3. Se Pu. 2025 Nov;43(11): 1222-1234
    [Determination of 51 per- and polyfluoroalkyl substances in raw water and drinking water by online solid-phase extraction coupled with ultra performance liquid chromatography- tandem mass spectrometry].
    Yong-Yan Chen, Jia Lyu, Bi-Xiong Ye, Lan Zhang, Yuan-Yuan Wang, Ning Jin.
      In this study， an online solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry （online SPE-UPLC-MS/MS） method was established to rapidly screen and determine 51 per- and polyfluoroalkyl substances （PFASs） in raw and drinking water. Ammonium formate and 24 PFAS internal standards were added to each sample. The ammonium formate concentration in the sample was 2 mmol/L after mixing， and the contents of the PFAS internal standards ranged between 2.5 and 50 ng/L. Each sample was filtered through a 0.22-μm cellulose acetate filter， and a 5-mL aliquot was injected and adsorbed using an HLB online solid-phase extraction column and rinsed with 2 mmol/L ammonium formate. Acetonitrile and 2 mmol/L aqueous ammonium formate were used as mobile phases. Separation was performed on a BEH C18 chromatographic column with gradient elution. Electrospray ionization source negative ion mode and multiple reaction monitoring mode were used for detection， with quantification performed using the internal standard method. The method was validated by determining accuracies and precisions for the 51 PFASs using raw water and drinking water as matrices. Excellent linear relationships within their respective ranges were observed， with correlation coefficients （r2）>0.995. The method exhibited limits of detection （LODs， S/N=3） and quantification （LOQs， S/N=10） of 0.03-1.5 and 0.1-5.0 ng/L， respectively. The PFASs were spiked at levels of 1， 10 and 50 ng/L， with spiked recoveries of 60.2%-126.9% and 60.4%-122.6% obtained for raw- and drinking-water samples， respectively， along with corresponding relative standard deviations （RSDs， n=6） of 0.3%-17.9% and 0.4%-17.7%， respectively. The developed method was used to determine PFAS residues in raw and drinking water， with perfluoroalkyl carboxylic acids， perfluoroalkyl sulfonic acids， and perfluoroalkyl ether acids detected at relatively high rates with contents of 0.1-209.7 and 0.1-63.6 ng/L in raw water and drinking water， respectively. Compared to offline solid-phase extraction methods， the developed method requires fewer samples， is more convenient from a sample-collection perspective， and uses lower quantities of internal standards. It also analyzes rapidly and is highly sensitive and reproducible. Only 20 min was required to determine the 51 PFASs at the ng/L level， from online enrichment to detection. The developed method is suitable for the trace determination of various types of PFAS， such as perfluoroalkyl carboxylic acids， perfluoroalkyl sulfonic acids， perfluoroalkyl ether acids， fluorotelomers， and fluoroalkyl sulfonamides， in raw and drinking water， thereby effectively boosting the detection efficiency for perfluoroalkyl substances in water in a manner that has substantial practical applications value.
    Keywords:  drinking water; online solid-phase extraction （online SPE）; per- and polyfluoroalkyl substances （PFASs）; raw water; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.12020
  4. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Oct 28. pii: S1570-0232(25)00382-4. [Epub ahead of print]1268 124828
    Impact of Preprocessing and data analysis strategies on metabolite annotation in biological samples: A mass spectrometry-based metabolomics study using feature-based molecular networking.
    Qinyu Xiao, Jiamin Yang, Jianjun Xie, Qing Liu, Hongbo Huang, Yiwen Tao, Bo Ding.
      Metabolomics workflows involve multiple complex steps including sample collection, storage, preparation, metabolite extraction, analytical platforms selection, data acquisition and interpretation. Each step may introduce variability that affects the quality and reliability of metabolomic data. To systematically investigate the effects of these factors on metabolomics outcomes, plasma samples from four different anatomical sites of colon cancer patients were analyzed using Liquid chromatography- Quadrupole-Exactive Orbitrap mass spectrometry (LC-Q-Exactive Orbitrap MS) for untargeted metabolomics. Response surface methodology was employed to optimize the ultrasound-assisted extraction conditions during sample pretreatment. Data analysis strategies were systematically evaluated, including Feature-Based Molecular Networking (FBMN) construction parameters and comparative assessment of different FBMN platforms for metabolite annotation. The optimized extraction conditions were determined as 300 % methanol concentration, sample freezing at -20 °C for 40 min, followed by ultrasonication for 5 min. Sample standardization protocols requiring single-use portioning and limiting freeze-thaw cycles to ≤2-3 cycles were identified as essential for reliable biomarker discovery and therapeutic mechanism exploration. Optimal FBMN construction parameters comprised a 25-min gradient elution time, 50 mm chromatographic column length, and high sample concentration. Comparative evaluation of Global Natural Products Social Molecular Networking (GNPS) and MZmine implementations of FBMN revealed that GNPS was recommended for studies prioritizing comprehensive annotation coverage and discovery-oriented metabolomics, while MZmine was preferred for method development, or applications requiring local processing without external data upload. This study demonstrated that preprocessing and data analysis strategies were critical determinants of data quality in untargeted plasma metabolomics. The findings provided evidence-based recommendations for experimental design, storage conditions, and data handling procedures that can guide protocol standardization and minimize undesired analytical variation in metabolomics studies.
    Keywords:  FBMN; GNPS; MZmine; Mass spectrometry-based metabolomics; Metabolite annotation; Sample preprocessing
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124828
  5. J Sep Sci. 2025 Nov;48(11): e70317
    Development and Clinical Validation of a Simultaneous Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry Assay for Three Triazole Antifungal Agents and Two Metabolites Enabling Precision Therapeutic Drug Monitoring in Chinese Patients With Hematologic Malignancies.
    Mingjie Yu, Fang Liu, Lirong Xiong, Qing Dai, Lin Cheng, Yongchuan Chen.
      Triazole antifungals are clinically established agents for the prophylaxis and treatment of invasive fungal diseases in patients with hematologic malignancies. To address the requirements of clinical therapeutic drug monitoring, we developed and validated a novel ultra-high-performance liquid chromatography tandem mass spectrometry method for the simultaneous quantification of three triazole antifungals and their metabolites in human plasma: itraconazole (ICZ), hydroxy-itraconazole (ICZ-OH), voriconazole (VCZ), voriconazole N-oxide (VCZ N-oxide), and posaconazole. The five target analytes were successfully separated using a BEH C18 column (2.1 × 50 mm, 1.7 µm) maintained at 40°C, with gradient elution employing mobile phase A (5.0 mM ammonium acetate in water containing 0.1% formic acid) and mobile phase B (100% acetonitrile [ACN]) at a constant flow rate of 0.4 mL/min. A positive ion pattern was chosen for quantification under multiple reaction monitoring. Following the addition of 10 µL of internal standard, 50 µL of plasma underwent protein precipitation with ACN, and the resulting supernatant was diluted for subsequent analysis. Method validation followed Food and Drug Administration guidelines and Chinese Pharmacopoeia regulations, demonstrating acceptable accuracy, precision, matrix effects, recovery, and stability. The calibration curves exhibited excellent linearity over the range of 0.1-10 µg/mL for all five analytes, with correlation coefficients (r2) ≥ 0.9962, while the lower limit of quantification and limit of detection were established at 0.1 and 0.03 µg/mL, respectively. The intra- and inter-day coefficients of variation were below 8.7% at all concentration levels, and the accuracy was 90.0%-113.0%. This methodology was successfully applied for TDM of three triazole antifungals and their metabolites in 150 patients with hematologic malignancies. Therefore, the method demonstrates good analytical performance, establishing its reliability for clinical TDM of triazole antifungals.
    Keywords:  LC‐MS/MS; TDM; hematologic malignancies; metabolites; triazole antifungals
    DOI:  https://doi.org/10.1002/jssc.70317
  6. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Oct 26. pii: S1570-0232(25)00392-7. [Epub ahead of print]1268 124838
    Validation of a highly sensitive assay for the determination of rivastigmine in human plasma for pharmacokinetic studies.
    Maksim Dolov, Igor Rodin.
      A fast and highly sensitive LC-MS/MS method has been developed and validated for measuring rivastigmine levels in human plasma. The extraction of rivastigmine from plasma was performed using a simple and quick protein precipitation technique (PPT). The internal standard used was atazanavir-d5. Chromatographic separation was achieved using a YMC-Triart C18 column, followed by detection with mass spectrometry. The mass transitions monitored were m/z 251.1 > 206.0 for rivastigmine, and m/z 710.5 > 168.1 for atazanavir-d5. This method involves rapid plasma extraction, straightforward gradient chromatography, and mass spectrometric detection, allowing for the detection of rivastigmine at sub-nanogram per milliliter levels. The method was validated over a linear range of 25 to 5000 pg/ml, with a correlation coefficient of at least 0.9980. Both intra- and inter-day precision and accuracy were within 15 %. The overall recovery rates for rivastigmine and atazanavir-d5 were close to 100 %. The total analysis time per sample was only 3 min. This method was successfully applied to determine the pharmacokinetic parameters of rivastigmine after a single oral dose of 1.5 mg (capsules) in 26 healthy volunteers.
    Keywords:  LC/MS-MS; Rivastigmine; pharmacokinetics; validation
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124838
  7. Clin Chem Lab Med. 2025 Nov 10.
    An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of mycophenolic acid glucuronide in human serum and plasma.
    Tobias Schierscher, Janik Wild, Neeraj Singh, Martina Bachmann, Julia Hoop, Friederike Bauland, Andrea Geistanger, Lorenz Risch, Christoph Seger, Judith Taibon.
       OBJECTIVES: A candidate reference measurement procedure (RMP) based on isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated to accurately measure serum and plasma concentrations of mycophenolic acid glucuronide (MPAG).
    METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was utilized for determining the absolute content (mass fraction; g/g) of the reference material, thereby, establishing the traceability to SI units. Separation of MPAG from potential interferences, whether known or unknown, was accomplished by using a Phenomenex Luna C18(2) column. For sample preparation, a protocol based on protein precipitation followed by a high-dilution step was established. A multi-day validation experiment evaluated precision and accuracy. Reproducibility was determined by comparing the results of the procedure between two independent laboratories. Measurement uncertainty (MU) was assessed in accordance with current guidelines.
    RESULTS: The RMP demonstrated high selectivity and specificity enabling the quantification of MPAG in the range between 0.750 and 600 μg/mL. The intermediate precision and repeatability (n=60, measurements) were found to be in the range from 0.9 to 3.7 % for serum samples and from 1.2 to 4.6 % for plasma samples. The repeatability was less than 3.5 % for serum samples and less than 4.0 % for plasma samples. The relative mean bias ranged from -0.9 to 3.2 % for serum samples and from -0.3 to 2.9 % for plasma samples. The expanded measurement uncertainties (k=2) for single measurements ranged between 2.4 and 7.7 % and were further reduced performing a target value assignment (n=6) resulting in expanded measurement uncertainties between 1.8 and 3.3 % (k=2), respectively.
    CONCLUSIONS: We herein present a new LC-MS/MS-based candidate RMP for MPAG in human serum and plasma which offers a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.
    Keywords:  ID-LC-MS/MS; mycophenolic acid glucuronide; qNMR; reference measurement procedure; standardization; traceability
    DOI:  https://doi.org/10.1515/cclm-2025-1052
  8. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Oct 29. pii: S1570-0232(25)00394-0. [Epub ahead of print]1268 124840
    An LC-MS/MS method for simultaneous quantification of pyrotinib, docetaxel and paclitaxel in human plasma and its application to therapeutic drug monitoring.
    Lingxiao Zhang, Jie Qu, Xinyan Sun, Haiyan Dong, Hongping Yao, Xiaoliang Cheng.
      Pyrotinib which is a novel irreversible tyrosine kinase inhibitor plus docetaxel or paclitaxel is effective for patients with Her2 positive early or advanced breast cancer including those who failed in first-line treatment. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and verified for simultaneous quantification of pyrotinib, docetaxel and paclitaxel in human plasma, and applied to therapeutic drug monitoring. A reversed-phase Hypersil GOLD aQ column eluted by a gradient mobile phase composed of water and acetonitrile both containing 0.1 % formic acid under flow rate of 0.3 mL min-1 was used for chromatographic separation. The mass spectrometry was operated in positive electrospray ionization mode, and selective reaction monitoring was applied for quantitative analysis. With imatinib as internal standard, one-step deproteinization approach with acetonitrile was applied to extract analytes and purify specimens. This method was adequately validated according to guidelines in terms of specificity and selectivity, sensitivity, linearity, extraction recovery, matrix effect, precision and accuracy, dilution integration and stability. The validated method was applied to therapeutic drug monitoring for breast cancer patients receiving pyrotinib and taxanes based chemotherapy. The therapeutic drug monitoring results showed that the plasma concentration of pyrotinib, docetaxel and paclitaxel varied significantly among individuals. Therapeutic drug monitoring for pyrotinib, docetaxel and paclitaxel is essential for individualized treatment to ensure efficacy and safety.
    Keywords:  Docetaxel; LC-MS/MS; Paclitaxel; Pyrotinib; Therapeutic drug monitoring
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124840
  9. Clin Chem Lab Med. 2025 Nov 04.
    An isotope dilution-liquid-chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of total and free valproic acid in human serum and plasma.
    Tobias Schierscher, Linda Salzmann, Neeraj Singh, Sandra Fleischer, Carina Schäfer, Julia Hoop, Friederike Bauland, Andrea Geistanger, Lorenz Risch, Christoph Seger, Judith Taibon.
       OBJECTIVES: An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (cRMP) was developed and validated to measure serum and plasma concentrations of the total and free form of valproic acid.
    METHODS: Quantitative nuclear magnetic resonance spectroscopic methodology was used to determine the absolute content (g/g) of the reference material, ensuring traceability to SI units. Separation of valproic acid from potential unknown interferences was achieved with reversed-phase chromatography. A protein precipitation protocol was established for sample preparation for total valproic acid, while the free form was separated by ultrafiltration. Assay validations and measurement uncertainties were aligned with guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement.
    RESULTS: The cRMPs were highly selective and specific with no evidence of matrix effects, allowing quantifying total and free valproic acid in a range of 2.40-145 μg/mL and 1.60-42.0 μg/mL, respectively. Intermediate precision was <4.0 % and repeatability CV ranged from 0.9 to 3.5% for all concentrations of free and total valproic acid. The relative mean bias ranged from -0.4 to 4.1 % for native serum and from -0.3 to 3.5 % for Li-heparin plasma levels for total valproic acid. Free valproic acid showed mean biases between -2.9 and 4.0 % for native serum and ultrafiltrates. Measurement uncertainties for single measurements and target value assignment ranged from 1.7 to 3.4 % and 0.9-1.3 %, respectively, for total valproic acid. Free valproic acid ranged from 2.0 to 4.1 % and from 0.8 to 1.5 % for single measurements and target value assignment, respectively.
    CONCLUSIONS: We present novel ID-LC-MS/MS-based cRMPs for total and free valproic acid in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.
    Keywords:  free valproic acid; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR; reference measurement procedure; traceability; valproic acid
    DOI:  https://doi.org/10.1515/cclm-2025-1053
  10. Anal Bioanal Chem. 2025 Nov 01.
    Comparative evaluation of comprehensive offline 2D-LC strategies coupled to MS for untargeted metabolomic studies of human urine.
    Maria Grübner, Andreas Dunkel, Frank Steiner, Thomas Hofmann.
      Out of the broad selection of analytical methods applied in metabolomic studies, liquid chromatography coupled to mass spectrometry (LC-MS) has the highest coverage potential. In that regard, the quality of the separation process is crucial for the analytical outcome. Reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) are widely applied, and the potential of various setups to combine these modes for more complementary data has been deeply explored. In our previous study on the orthogonality of LC conditions in the field of metabolomics, combinations of a mixed-mode phase with parallel RP and ion-exchange (IEX) properties and several HILIC columns exhibited the widest compound distributions in a two-dimensional (2D) separation space. For further performance evaluation, an offline comprehensive 2D-LC-TOF-MS (LC×LC-TOF-MS) system was set up with the mixed RP/IEX mode in the first dimension (1D) and HILIC mode in the second dimension (2D). The transfer of fractions to the HILIC column and the effect of offline fraction preparation procedures (dilution and evaporation approaches) were comparatively investigated by using reference substances. In addition, the separation performance of the offline LC×LC-TOF-MS system with and without offline fraction preparation was assessed in comparison to other common LC-TOF-MS strategies (direct flow injection DFI, 1D-LC, serial coupling LC) by the number of detectable features in a human urine sample. In conclusion, the direct transfer of 5 µL fraction volumes without offline treatment was the most promising approach for future application in untargeted metabolomic studies for marker identification from human urine.
    Keywords:  Comprehensive offline two-dimensional LC; Evaporation; Fractionation; HILIC; Metabolomics; Mixed-mode chromatography
    DOI:  https://doi.org/10.1007/s00216-025-06195-2
  11. PLoS One. 2025 ;20(11): e0335264
    A Fragmentation behavior-guided UHPLC-Q-Orbitrap HRMS method for the quantitative analysis of 26 perfluoroalkyl substances and their alternatives in water.
    Guojian Shao, Tao Liu, You Xia, Peng Zhang, Ye Wang.
      Perfluoroalkyl substances (PFASs), including newly introduced alternatives, are of global concern due to their environmental persistence, bioaccumulation, and potential health risks. We developed and validated a sensitive, selective UHPLC-Q-Orbitrap HRMS method for the comprehensive analysis of 26 PFASs-including perfluorocarboxylic acids (PFCAs), perfluorosulfonic acids (PFSAs), and emerging substitutes-in diverse water matrices. Sample preparation used WAX solid-phase extraction with isotope-labeled internal standards and separation on a BEH C18 column; quantitative performance was assessed under Full MS, targeted SIM, and PRM. The method achieved excellent linearity, sub-ng/L detection capability, and robust recoveries and precision across matrices, with PRM offering the best balance of sensitivity and selectivity. Characteristic fragmentation patterns, like decarboxylation and desulfonation, supported structural confirmation and resulted in diagnostic fragments that facilitated isomer differentiation and increased the reliability of identifying low-abundance compounds. This fragmentation-guided UHPLC-HRMS approach enables high-confidence detection and quantification of both legacy and novel PFASs and provides a practical tool for environmental monitoring and risk assessment.
    DOI:  https://doi.org/10.1371/journal.pone.0335264
  12. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Sep 15. pii: S1570-0232(25)00347-2. [Epub ahead of print]1268 124793
    Method development and validation for quantitative determination of urinary biomarkers of food intake for multiple foods.
    Christoph Hassenberg, Sebastian T Soukup, Marina Armeni, Achim Bub, Jil V Cannas, David Fuentes, Otto Savolainen, Stephanie Seifert, Rikard Landberg, Sabine E Kulling, Carina I Mack.
      Biomarkers of food intake (BFIs) have emerged as a promising objective tool to complement traditional self-reported dietary assessment in nutritional research, with the potential to reduce systematic errors and improve accuracy. The development of comprehensive and robust quantification methods for BFIs is essential for widespread application. However, existing methods typically cover only a moderate number of BFIs per method, hindering their wide application in the field. In this study, we present the development and validation of a method for simultaneous quantification of 80 BFIs in urine reflecting 27 foods. The method utilizes a simple sample preparation procedure, followed by separation using both high-performance liquid chromatography (HPLC) on a C18 column and a hydrophilic interaction chromatography (HILIC) column, combined with tandem mass spectrometry in positive and negative mode (HPLC-MS/MS) (individual runs: 6 min). The working range for each analyte was determined in urine samples from a non-randomized, non-blinded nutritional intervention study. The method was validated with respect to selectivity, linearity, robustness, matrix effects, recovery, accuracy, and precision. In total, 44 BFIs could be absolutely quantified without or with only limitations at low concentrations, while 36 BFIs could only be measured semi-quantitatively, including 16 BFIs with limited validation data due to uncertainties. The 80 BFIs represent 27 foods (6 semi-quantitative) frequently consumed in European diets, including 24 plant-derived and 3 animal-derived items. The future implementation of this large-scale BFI quantification method in nutritional studies is expected to demonstrate the benefits of routinely measuring BFIs to improve the accuracy of dietary assessment.
    Keywords:  Biological sample; Biomarker of food intake; Dietary assessment; HPLC-MS/MS; Method validation; Multi-target; Quantification
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124793
  13. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Oct 26. pii: S1570-0232(25)00390-3. [Epub ahead of print]1268 124836
    Development and validation of an LC-MS/MS method for Tirzepatide, a dual GIP/GLP-1 receptor agonist, in rat plasma for application to a pharmacokinetic study.
    Hae-In Choi, Hyeon-Cheol Jeong, Jong-Woo Jeong, Jaeyoung Lee, Da Hae Kim, Kyong-Cheol Ko, Yoon-Jee Chae, Kyeong-Ryoon Lee.
      A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of tirzepatide, a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors in rat plasma. Tirzepatide was extracted from rat plasma by protein precipitation using methanol. Chromatographic separation was achieved using a peptide C18 column with gradient elution of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed in positive electrospray ionization mode using multiple reaction monitoring with transitions of m/z 1204.4 → 1473.6 for tirzepatide and m/z 1029.4 → 1238.4 for the internal standard, semaglutide. The developed method exhibited good linearity over a concentration range of 1-1000 ng/mL (r2 > 0.99). Intra- and inter-day accuracy (-4.324-5.057 %) and precision (5.250-9.000 %) met the regulatory criteria at all quality control levels, and were stable under various plasma handling and storage conditions. The validated method was successfully applied to a pharmacokinetic study in rats following the intravenous and subcutaneous injection of tirzepatide at 0.3 mg/kg. The terminal half-lives were 10.04 h and 9.803 h after intravenous and subcutaneous administration, respectively, indicating comparable elimination profiles. The bioavailability following subcutaneous dosing was estimated to be approximately 62.38 %. These findings highlight the robustness and applicability of the developed method, suggesting its potential utility for the quantitative analysis of other peptide therapeutics with structures or mechanisms of action similar to those of tirzepatide.
    Keywords:  Bioanalysis; Dual GIP/GLP-1 receptor agonist; LC-MS/MS; Pharmacokinetics; Tirzepatide; Validation
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124836
  14. Yakugaku Zasshi. 2025 ;145(11): 877-881
    [Development of Enantioselective Imaging Method for Chiral Molecules by on-Tissue Chiral Derivatization and Ion Mobility Mass Spectrometry].
    Eiji Sugiyama.
      Bioactive molecules are mostly chiral, and their enantiomers often exhibit different biological properties, including pharmacological effects and toxicity. Recent biochemical studies have revealed that certain trace-level chiral metabolites are associated with specific pathological conditions, including chronic kidney disease and metabolic disorders. This finding highlights the importance of enantioselective imaging techniques that can help visualize the spatial distribution and dynamic behavior of individual enantiomers. Although enantioselective biochemical analyses, such as those based on chromatography or electrophoresis, have proven effective in separating enantiomers, progress in enantioselective imaging methods has been limited. Ion mobility spectrometry/mass spectrometry imaging (IMS/MSI) has emerged as a powerful tool with the potential to enable the enantioselective imaging of minor chiral metabolites. However, suitable chemical structures that can achieve both sufficient resolving power for the target enantiomer in IMS and high ionization efficiency in MSI remain unclear. This review highlights the development of enantioselective imaging methods based on on-tissue chiral derivatization and IMS/MSI. After exploring suitable chiral derivatization reagents, we designed a new charged chiral tag that enabled complete separation of a pair of enantiomers by IMS, sensitive detection of D,L-2-hydroxyglutaric acid by mass spectrometry, and the visualization of their distribution in the mouse testis by IMS/MSI. This approach can be further expanded to analyze other chiral molecules and has great potential for unveiling the enantioselective distribution and dynamics of minor chiral metabolites in biological tissues.
    Keywords:  chiral; derivatization; imaging; ion mobility spectrometry; mass spectrometry
    DOI:  https://doi.org/10.1248/yakushi.25-00128
  15. Se Pu. 2025 Nov;43(11): 1244-1251
    [Quantitation of cow's milk allergen in hypoallergenic infant formulas using ultra-high performance liquid chromatography- tandem mass spectrometry].
    Dao-Kun Xu, Yang Yang, Hao-Lun Huang, Xiao-Jie Sun, Wen-Yan Hu, Xin-Mei Liu, Jun Yang.
      Cow's milk protein allergy （CMPA） has become a common public health concern and hypoallergenic formula （partially or extensively hydrolyzed formula） is considered as an alternative during infancy. Alcalase and papain are widely used in industrial production of hydrolysates， as they can modify or destroy the linear and conformational epitopes of cow's milk allergens （CMAs）， thus leading to decreased allergenicity. However， despite considerable emphasis has been laid on the manufacturing technique and nutritional value of hypoallergenic formulas， whether there are traces of CMAs in hypoallergenic formulas， remains unknown. Consequently， the need for a quantitative method of detecting multiple CMAs at a time is rising. Traditionally， enzyme-linked immunosorbent assay （ELISA）-based methods have proved to be advantageous with a wide availability of commercial kits for CMAs detection. Moreover， polymerase chain reaction （PCR）， as one of DNA-based methods， has shown exceptional specificity and high sensitivity. However， they both may encounter false-positive results and high-throughput detection is challenging. In the past two decades， mass spectrometry （MS）-based proteomic technology has advanced， including improvements in computational capability， algorithm model， as well as mass spectrometric platforms. MS is gaining more and more attention in the field of food allergen detection， allowing multiplex allergen detection with high sensitivity， accuracy and selectivity in a single run. In this study， an analytical method was developed for the simultaneous quantitation of six CMAss （namely， α-lactalbumin， β-lactoglobulin， αS1-casein， αS2-casein， β-casein， and κ-casein） in hypoallergenic formulas using ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）. Proteins of spiked hypoallergenic formula were extracted with ammonium bicarbonate and digested with trypsin. After digestion， the samples were purified with a C18 spin column， and analyzed by UHPLC-MS/MS （AB SCIEX Triple Quad 6500+）. Separation was achieved on a Waters ACQUITY UPLC Peptide CSH C18 column （150 mm×2.1 mm， 1.7 μm）. The mobile phase consisted of 0.1% formic acid aqueous solution and 0.1% formic acid in acetonitrile， and the flow rate was 0.3 mL/min. The sample injection volume was 10 μL， and the column temperature was set to 40 ℃. Mass spectrometer was operated in positive electrospray ionization （ESI+） mode， and multiple reaction monitoring （MRM） mode was adopted. Precursor ions and product ions of marker peptides were predicted by the Skyline software. Mass spectrometric parameters such as declustering potential （DP） and collision energy （CE） were also optimized. Afterwards， by taking allergen epitopes and mass spectrometric responses into consideration， six peptides were retained for quantitative analysis. Calibration curves were constructed with good linearity in the range of 0.05-500 mg/kg， and correlation coefficients （r2） greater than 0.99. The limits of detection （LODs） were 0.05-5.0 mg/kg， and limits of quantification （LOQs） were 0.1-10.0 mg/kg， respectively. The recoveries of the developed method in incurred hypoallergenic formulas ranged from 74.8% to 93.4%. The intraday and interday accuracies ranged from -25.09% to -6.09% and from -24.0% to -5.41%， with precisions between 1.08%-5.05% and 1.35%-4.85%， respectively. Screening of commercial hypoallergenic formula samples confirmed existence of CMAs in three partially hydrolyzed formulas （pHF） and two extensively hydrolyzed formulas （eHF）， with contents ranging from 0.48 to 27.36 mg/kg. The analytical method proposed in this study exhibited high sensitivity， high-throughput capability， and can be used to monitor traces of CMAs in hypoallergenic formulas.
    Keywords:  cow’s milk allergens （CMAs）; hypoallergenic infant formula; peptides， label-free quantification （LFQ）; ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.12030
  16. Anal Methods. 2025 Nov 06.
    Automated dual-cartridge solid-phase extraction method for multi-residue determination of veterinary drugs in bovine muscle, liver, fat, and milk.
    Ryu Mochizuki, Shizuka Saito-Shida, Maki Saito, Takaaki Taguchi, Tomoaki Tsutsumi.
      Solid-phase extraction (SPE) is a widely used sample preparation technique for the determination of veterinary drug residues in foods due to its effectiveness in removing matrix components. However, conventional SPE methods are often time consuming and labor intensive. In this study, we developed an automated sample preparation method employing a dual-cartridge SPE system for the multi-residue determination of veterinary drugs in bovine-derived food matrices. The method integrates two C18 cartridges connected in series, with controlled water addition between cartridges to improve cleanup efficiency. Analytical performance was evaluated for 52 veterinary drugs in bovine muscle, liver, fat, and milk at a concentration of 0.01 mg kg-1. Minimal matrix effects were observed, allowing for accurate quantification using solvent-based calibration without the need for matrix-matched standards or isotope-labeled internal standards. Satisfactory analytical performance was obtained for approximately 80% of the analyte-matrix combinations, with trueness values ranging from 70% to 120% and intra- and inter-day precision values within 25% and 30%, respectively, although some combinations fell outside these criteria due to degradation or matrix effects. The method also demonstrated high selectivity, with no interfering peaks detected near the retention times of target analytes. Overall, the developed automated SPE method provides a robust and reliable platform for quantification of veterinary drug residues in complex bovine-derived food products, supporting its suitability for use in routine food safety monitoring and regulatory surveillance.
    DOI:  https://doi.org/10.1039/d5ay01515d
  17. Commun Chem. 2025 Nov 04. 8(1): 326
    A large language model for deriving spectral embeddings for accurate compound identification in mass spectrometry.
    Yang Xu, Yixiao Ma, Weijie Xu, Zuliang Yang, Kai Ming Ting.
      Identifying chemical components in complex mixtures is a crucial task across many scientific disciplines. Mass spectrometry serves as a key analytical tool for this purpose, yet the accurate identification of compounds from their spectra remains a major bottleneck. Here we introduce LLM4MS, a method that leverages the latent expert knowledge within large language models to generate discriminative spectral embeddings for improved compound identification. LLM4MS is designed to incorporate potential chemical expert knowledge, enabling accurate matching. Evaluated against a million-scale open-source in-silico library using the NIST23 library as a test set, LLM4MS achieves a Recall@1 accuracy of 66.3% (and a Recall@10 accuracy of 92.7%), representing a 13.7% improvement over the state-of-the-art Spec2Vec. Furthermore, LLM4MS enables ultra-fast mass spectra matching, achieving a speed of nearly 15,000 queries per second. Thus, LLM4MS opens up avenues to significantly enhance compound identification in mass spectrometry and accelerate chemical discovery.
    DOI:  https://doi.org/10.1038/s42004-025-01708-7
  18. Sci Rep. 2025 Nov 04. 15(1): 38460
    Integrating mass spectrometry and hyperspectral imaging for protoporphyrin IX detection in malignant glioma tissue.
    Fabio D'Alessandro, Anna Walke, Nora Maren Kiolbassa, Walter Stummer, Simone König, Eric Suero Molina.
      Maximal safe tumor resection is crucial for the treatment of high-grade gliomas (HGG). 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery enhances tumor visualization by inducing protoporphyrin IX (PpIX) accumulation. However, current fluorescence-based observation devices lack the sensitivity for detecting tumor cells in low-density infiltrative zones. Hyperspectral imaging (HI) offers a potential solution. In this study, HI-derived PpIX measurements were compared to those obtained from reversed-phase liquid chromatography coupled to mass spectrometry (LC-MS), a method that delivers accurate concentrations. Additionally, we investigated coproporphyrins (Cp) I and III, since they potentially interfere with PpIX determination. Pig brain was used as a surrogate for protocol development and acquisition of comparative HI and LC-MS reference data, which were subsequently used to evaluate the results obtained from 27 biopsies from nine patients undergoing 5-ALA-mediated tumor resection. During sample preparation for LC-MS, 80% PpIX and 45% combined Cp I & III were recovered from brain tissue. For LC-MS quantification of PpIX, accuracy ranged from 98 to 137%, and coefficient of variation was 5-14%, indicating sufficient precision. For HI, the values were 77-121% and 11-31%, respectively. Notably, HI significantly overestimated PpIX concentrations compared to those determined by LC-MS. This study highlights LC-MS as a reliable method for porphyrin quantification and suggests that HI workflows need further optimization for accurate tumor delineation in HGG.
    DOI:  https://doi.org/10.1038/s41598-025-26245-0
  19. Se Pu. 2025 Nov;43(11): 1235-1243
    [Determination of eight per- and polyfluoroalkyl substances in seawater using dispersive membrane extraction-ultra-high performance liquid chromatography-tandem mass spectrometry].
    Zhen Ren, Ji-Ping Ma, Ya-Jing Liu, Lan Zhang, Ge-Ge Wu, Shuang Li.
      Per- and polyfluoroalkyl substances （PFASs） are widely utilized in various industrial applications. Their persistence in ecosystems raises significant global environmental health concerns. Numerous studies have confirmed the toxicity of PFASs to human endocrine and immune systems. The carcinogenic risks associated with PFASs exposure increasingly alarm public health authorities worldwide. As a result， regulatory policies have been implemented to restrict both the production and environmental release of PFASs. In China， the GB 5749-2022 standard establishes stringent limits for PFASs concentrations in drinking water， capping levels of perfluorooctane sulfonic acid （PFOS） at 40 ng/L and perfluorooctanoic acid （PFOA） at 80 ng/L. Effective monitoring of PFASs requires advanced analytical techniques that exhibit exceptional sensitivity. Ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） has emerged as the preferred technique for detecting these substances due to its superior selectivity and low detection limits. However， complex environmental matrices necessitate optimized sample pretreatment strategies to enhance analysis accuracy. Efficient extraction methods must effectively address matrix interference while ensuring analyte enrichment. Dispersive membrane extraction （DME） presents distinct advantages for the pretreatment of PFASs； its operational simplicity is complemented by high enrichment capabilities and efficient mass transfer. The selection of extraction materials critically influences DME performance parameters. Metal-organic frameworks （MOFs） are porous materials composed of organic ligands and metal clusters， featuring tunable pores， high surface areas， and rapid mass transfer. These unique properties enable their current use in sample pretreatment for environmental analysis. This study integrates DME with UHPLC-MS/MS utilizing cationic MOF membranes， enabling simultaneous detection of eight different PFASs within seawater matrices. Optimized parameters ensure reliable quantification of trace-level contaminants throughout this process. The experimental design assessed various parameters， including types of organic solvents （methanol and acetonitrile）， ammonium acetate concentrations in the aqueous phase （0.1， 0.5， 1.0， 2.0， and 5.0 mmol/L）， as well as ion source voltages （-2 500， -3 500， and -4 500 V）， all of which influenced detection efficacy. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column （100 mm×2.1 mm， 1.7 μm）. Gradient elution combined 1 mmol/L ammonium acetate with acetonitrile for optimal results. Detection was conducted utilizing electrospray ionization （ESI） in negative ion scanning mode coupled with multiple reaction monitoring （MRM）. The results indicated that all eight PFASs could be effectively separated within a timeframe of 12 min， exhibiting favorable peak shapes and high response values. Under optimal conditions， the eight PFASs demonstrated strong linearity across their respective concentration ranges； the correlation coefficients （r²） were all not less than 0.990 7. Recoveries of PFASs at spiking levels of 10， 50， and 100 ng/L ranged from 50.4% to 116.4%， while intra-day and inter-day relative standard deviations （RSDs） varied from 1.0% to 19.2% and from 2.2% to 19.5%， respectively. The method's limits of detection （LODs） ranged from approximately 0.07 ng/L to 0.49 ng/L， whereas the limits of quantification （LOQs） varied from around 0.22 ng/L to 1.63 ng/L. Jiaozhou Bay serves as a typical semi-enclosed bay along the Pacific coast where terrestrial runoff converges nutrients alongside diverse organic/inorganic pollutants that pose potential health risks for aquatic organisms inhabiting these waters. This method was utilized for the detection of the eight PFASs in the surface seawater of Jiaozhou Bay， and seven types were successfully identified. Among these， potassium 11-chloroeicosafluoro-3-oxaundecane-1-sulfonate （Minor F-53B） exhibited the highest detection concentration， with an average mass concentration of 17.11 ng/L. When compared to the 2018 detection results of PFASs in the surface seawater of Jiaozhou Bay， the average mass concentration of perfluorooctanoic acid （PFOA） has significantly decreased. Additionally， potassium 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate （F-53B）， one of the newly emerging PFASs， was detected in the surface seawater of Jiaozhou Bay， which may be attributed to the recent shift in PFASs production. The widespread use of these new PFASs may introduce environmental risks. These risks resemble those of traditional PFASs and require urgent attention. In summary， this method is operationally straightforward， rapid， and highly sensitive； it is suitable for analyzing the eight PFASs in seawater. This approach can provide a valuable data foundation and scientific basis for research and analysis concerning PFASs in environmental water bodies.
    Keywords:  cationic metal-organic framework; dispersive membrane extraction （DME）; per- and polyfluoroalkyl substances （PFASs）; ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.04009
  20. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Oct 22. pii: S1570-0232(25)00383-6. [Epub ahead of print]1268 124829
    Establishment of a LC-MS/MS method for the simultaneous quantitative determination of trimethylamine N-oxide and four precursors in human saliva.
    Jianyue Li, Bin Hu, Jie Wu, Dazhen Wang, Junyan Xia, Yanan Li, Yonghong Gao, Baorong Chen, Jianfeng Zhou, Zenghe Li.
      In this work, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantitatively detect the choline (CHL), trimethylamine N-oxide (TMAO), γ-butyrobetaine (GBB), betaine (BET), and ʟ-carnitine (CAR) in human saliva for the first time. Saliva samples collected from healthy volunteers were treated with acetonitrile for protein precipitation, followed by direct injection into the LC-MS/MS system for analysis. The method validation was performed in accordance with the CLSI C62-A standard guidelines. The linearity was in the range of 0.300-300.000 μmol/L for CHL, 0.005-5.000 μmol/L for TMAO, 0.050-50.000 μmol/L for GBB, 0.020-20.000 μmol/L for BET, and 0.100-100.000 μmol/L for CAR. The intra-day and inter-day precision ranged from 1.13 % to 8.34 % and from 1.63 % to 10.65 %, respectively. The recoveries and matrix effects of these analytes ranged from 94.31 % to 108.00 % and from 90.59 % to 111.37 %, respectively. The performance of the method met the requirements of the guidelines. The validated method was subsequently applied to analyze test saliva samples from 43 healthy volunteers and 4 coronary heart disease (CHD) inpatients, to evaluate the distributions of five analytes in healthy people and CHD inpatients. This study provides a reliable and accurate analytical approach for the clinical detection of five analytes in saliva.
    Keywords:  LC-MS/MS; Metabolism of trimethylamine N-oxide; Saliva; Trimethylamine N-oxide
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124829
  21. Anal Chem. 2025 Nov 06.
    Not One Method to Rule Them All: A Comparative Study of Chromatographic Platforms (RP-LC-, HILIC-, SFC-, and IC-HRMS) for Water Analysis.
    Jonathan Zweigle, Michael Schlüsener, Joana Flottmann, Tobias Bader, Nanna Hjort Vidkjær, Ulla E Bollmann, Jan H Christensen, Selina Tisler.
      Reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (RP-LC-HRMS) is the standard for nontarget screening (NTS) of environmental samples but lacks retention of highly polar contaminants. We compared 12 chromatographic methods across four platforms, RP-LC, anion chromatography (IC), supercritical fluid chromatography (SFC), and hydrophilic interaction chromatography (HILIC), using 127 environmentally relevant compounds (logDpH7.4 -5.6 to 6.6). Compounds were analyzed in solvent and for a polar subset, in groundwater enriched by vacuum evaporation. Data were collected across four laboratories using 5 RP-LC-, 3 HILIC-, 2 SFC-, and 2 IC-HRMS setups. Feature detection with standard tools yielded more false negatives for SFC and IC. To enable a fair and method-agnostic comparison, an extracted ion chromatogram (EIC)-based workflow was used. Of the 127 compounds, 125 were detected by at least one platform. For logDpH7.4 > 0, RP-LC covered ∼90%, followed by SFC (∼70%), while IC and HILIC each covered <30%. For very polar compounds (logDpH7.4 < 0), coverage dropped across all platforms. SFC and HILIC detected up to 60% of polar analytes; IC performed better in negative ionization mode, consistent with anion-exchange separation. Detection frequency declined with polarity, reflecting analytical limitations. Combining RP-LC with either SFC or HILIC increased coverage to 94%. In spiked groundwater, coverage was lower (73%) due to matrix effects and losses during vacuum enrichment. Peak widths were narrowest for SFC (∼2.5 s) and RP-LC (∼4 s) and broadest for HILIC (∼7 s) and IC (∼17 s). Retention times showed limited cross-platform correlation while ionization efficiency was consistent, except for SFC. As no single method provided full coverage, combining RP-LC with one complementary platform (SFC, HILIC, or IC) is required to extend chemical space in environmental NTS.
    DOI:  https://doi.org/10.1021/acs.analchem.5c04114
  22. Curr Opin Biotechnol. 2025 Oct 31. pii: S0958-1669(25)00117-X. [Epub ahead of print]96 103373
    Molecules to medicine: advances in metabolomics for natural product drug discovery.
    Victoria Deleray, Pieter C Dorrestein, Nicole E Avalon.
      Recent advances in metabolomics are accelerating natural product (NP) drug discovery. NPs possess diverse biological relevance and comprise a significant portion of our modern pharmacopeia. We highlight studies from the past two years with innovative discovery techniques, ranging from small sample analyses to large-scale data-driven approaches. We focus on nuclear magnetic resonance- and mass spectrometry-based metabolomics for their broad use and greatest advancements in the field. We highlight strategies that utilize computational tools to enable prioritization of samples based on structural novelty, cross-referencing structural data with bioactivity, and the development of innovative annotation techniques that surpass common library matching methods. We also look at the trajectory of metabolomic discovery of NPs over the last decade to inform how these platforms may further evolve. The goal is to enhance the likelihood and improve the efficiency of discovering NPs with pharmaceutical potential, while strategically harnessing data in order to reduce rediscovery and methodological redundancy.
    DOI:  https://doi.org/10.1016/j.copbio.2025.103373
  23. Anal Bioanal Chem. 2025 Nov 04.
    Exploring ammonium salt doping to enhance ion abundance for quantitative mass spectrometry imaging.
    Alora R Dunnavant, Seth M Eisenberg, David C Muddiman.
      Increasing ion abundance in mass spectrometry is essential for enhancing detection, quantification, and understanding of biomolecules involved in key cellular processes. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), MALDI, and nano-DESI have shown improved ion abundance when integrating specific concentrations of ammonium fluoride into the sample preparation and/or ionization steps. Herein, the importance of electronegativity and atomic size for the proposed mechanism of increased ion abundance is evaluated by testing a variety of ammonium halide salts. Ammonium fluoride was confirmed to result in the largest increase in ion abundance and was subsequently used to perform quantitative mass spectrometry imaging (qMSI) of glutathione (GSH) in healthy mouse liver tissue. Using ~70 µM NH4F as an ESI dopant, up to a ~ two-fold increase in ion abundance was observed for these biomolecules, as well as an improvement in the limit of detection, detection frequency, and quantification of endogenous GSH in tissue.
    Keywords:  Ammonium fluoride; Electrospray doping; Glutathione; IR-MALDESI; Quantitative mass spectrometry imaging
    DOI:  https://doi.org/10.1007/s00216-025-06198-z
  24. Se Pu. 2025 Nov;43(11): 1262-1267
    [Determination of genotoxic impurity in the bulk drug of crizotinib by high performance liquid chromatography-electrospray ionization tandem mass spectrometry].
    Xiao-Bing Zhan, Yi Qin, Ying Yan, Jie Zhou, Long-Shan Zhao.
      A method based on high performance liquid chromatography-electrospray ionization tandem mass spectrometry （HPLC-ESI-MS/MS） was developed for the determination of the potential genotoxic impurity （WHT1408-Q2H） in the bulk drug of crizotinib. An Agilent Eclipse XDB C8 chromatographic column （150 mm×4.6 mm， 3.5 µm） was used for chromatographic separation. The mobile phases were 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution at a flow rate of 0.4 mL/min. The column temperature was maintained at 40 ℃ and the sample size was 5 μL. In the mass spectrometry section， the electrospray positive ion （ESI+） mode with multiple reaction monitoring （MRM） scanning was adopted. The accurate mass of the ［M+H］+ parent ion of WHT1408-Q2H was m/z 205.3， and the accurate mass of the extracted fragment ion was m/z 121.0. The results of methodological validation demonstrated that the established method exhibited excellent specificity. The peak area and mass concentration of WHT1408-Q2H exhibited a good linear relationship within the range of 2-40 ng/mL， with a correlation coefficient （r） of 0.999 9. The limit of detection （LOD） and limit of quantitation （LOQ） for WHT1408-Q2H were 0.396 9 ng/mL and 1.984 6 ng/mL， respectively. The recoveries of WHT1408-Q2H at low， medium， and high levels were in the range of 95.6%-102.7%， while the relative standard deviations （RSDs） were between 0.4% and 0.7%. Finally， the proposed method was successfully applied to analyze three independent batches of the bulk drug of crizotinib. The results revealed that WHT1408-Q2H was not detected in all samples， indicating that the current production process can effectively control the content of this genotoxic impurity. In conclusion， the developed HPLC-ESI-MS/MS method is highly specific， sensitive， and simple， making it suitable for the stringent quality control of WHT1408-Q2H in the bulk drug of crizotinib. According to the M7 guideline on genotoxic impurities， this method is capable of accurately quantifying trace amounts of genotoxic impurities and will further ensure compliance with regulatory requirements and safeguard drug safety. Future applications may extend this analytical framework to similar genotoxic impurities assessment in other therapeutic compounds， thereby advancing the field of pharmaceutical impurity profiling and control.
    Keywords:  genotoxic impurity; high performance liquid chromatography-electrospray ionization tandem mass spectrometry （HPLC-ESI-MS/MS）; non-small cell lung cancer; the bulk drug of crizotinib
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.04017
  25. Anal Chem. 2025 Nov 07.
    Full-Signal Ultrahigh-Resolution NMR by Parameter Estimation.
    Simon G Hulse, Mathias Nilsson, Gareth A Morris, Mohammadali Foroozandeh.
      Pure shift NMR spectra, in which multiplet structure is suppressed, are widely used but exact a high price in sensitivity. Here we present CUPID (Computer-assisted Undiminished-sensitivity Protocol for Ideal Decoupling), which uses parametric estimation to produce pure shift NMR spectra from easily acquired 2D J-resolved (2DJ) data sets. Unlike previous practical methods for broadband pure shift NMR, it makes use of all of the available signal. CUPID is therefore effective even at sample concentrations where current methods are too insensitive to yield usable spectra. As an additional benefit, the estimation method used allows the extraction of individual multiplet structures from overlapping spectra. CUPID is freely available through NMR Estimation in Python (NMR-EsPy), an open-source package with a simple-to-use API, and comes with a graphical user interface that is accessible via Topspin, a widely used NMR software platform.
    DOI:  https://doi.org/10.1021/acs.analchem.5c03446
  26. Anal Bioanal Chem. 2025 Nov 01.
    A bio-SPME-LC-MS/MS method for the quantitative determination of cannabinoids in plasma samples: analytical strategy for optimization of matrix modification.
    Camila Marchioni, Emanuela Gionfriddo.
      Cannabis sp. has been widely used for both medicinal and recreational purposes globally. Analyzing cannabinoids and their metabolites in human plasma samples is crucial for managing acute intoxications and for therapeutic monitoring in patients. This study describes the optimization and validation of an innovative, sensitive, simple, and environmentally friendly biocompatible solid-phase microextraction (bio-SPME) method using a hydrophilic-lipophilic balance (HLB)/polyacrylonitrile (PAN)-thin film for the quantification of cannabinoids (cannabidiol, tetrahydrocannabinol, and cannabinol) and their main metabolites (11-hydroxy-Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol acid) in human plasma samples. Extraction efficiency was compared between two geometry SPME devices (e.g., thin film and fiber geometry), showing that the use of the thin film microextraction (TFME) device improved extraction performance. Matrix modification was optimized using a central composite design to determine the optimal combination of sample dilution, organic modifier addition, and salt usage. The most effective matrix modification was achieved with 380 µL of phosphate-buffered saline solution containing 0.015 mol L-1 sodium chloride and 57% acetonitrile added to 200 µL of human plasma. The extraction equilibrium time was established as 30 min, and the desorption step required only 20 min in 200 µL of desorption solvent. The method demonstrated linearity, high sensitivity, precision, and good accuracy, with no significant carryover between samples. When compared with recent methods, this approach scored higher in terms of "greenness" according to the AGREEprep metric, making it an environmentally sustainable option.
    Keywords:  Cannabinoids; DI-SPME; Matrix modification; Plasma samples; Thin film microextraction
    DOI:  https://doi.org/10.1007/s00216-025-06183-6
  27. Drug Metab Dispos. 2025 Oct 13. pii: S0090-9556(25)09492-9. [Epub ahead of print]53(11): 100183
    Pharmaceutical Metabolite Data Base, PharmMet DB: Reference data base for drug metabolites generated by human liver S9 fraction.
    Jennifer Jeon, Zachery R Jarrell, Choon-Myung Lee, Grant Singer, Jaclyn Weinberg, Ken H Liu, Edward T Morgan, Young-Mi Go, Dean P Jones.
      Drug monitoring is an essential component of precision therapeutics, yet existing data bases to support therapeutic monitoring are limited to data curated from the scientific literature or predicted in silico. We used human liver S9 fraction to generate metabolites from 1114 therapeutic drugs spanning diverse drug classes. Metabolites were analyzed by liquid chromatography-high-resolution mass spectrometry, annotated through differential analysis of preincubation and postincubation samples, curated by comparison to predicted metabolites from BioTransformer 3.0, and compiled into a human liver pharmaceutical metabolite resource, named "Pharmaceutical Metabolite Data Base (PharmMet DB)." Liquid chromatography-high-resolution mass spectrometry showed heterogeneity in product generation, with some drugs mostly being converted to predicted metabolites, while others were converted to hundreds of unpredicted products characterized by mass-to-charge ratio and chromatographic retention time. Phase I metabolism was dominant, with 30,752 oxidized drug metabolites. Glucuronidation was dominant for phase II metabolism, with 6311 drug metabolites. Notably, 89% of tested drugs produced at least 1 metabolite that was not predicted on BioTransformer 3.0, and these novel metabolites were most frequently detected for anti-inflammatory, central nervous system and antimicrobial drug classes. PharmMet DB provides experimental metabolite profiles to detect therapeutic drug exposures in human biospecimens without a requirement for prescription history. PharmMet DB usage with human epidemiology will advance pharmacometabolomics to improve understanding of drug efficacy, adverse reactions, and interactions in precision medicine. SIGNIFICANCE STATEMENT: Pharmaceutical Metabolite Data Base is a new data base of therapeutic drug metabolites suitable for use with liquid chromatography-high-resolution mass spectrometry to monitor patient adherence, detect unreported drug use, for example, in clinical trials, and enhance pharmacoexposomics and pharmacogenomics research. The data base was generated by incubation of therapeutic agents with human liver S9 fraction and curated relative to in silico predicted metabolites. Associated metadata for metabolic processes and drug classes enhance utility for clinical use, especially with untargeted metabolomics analyses of human samples.
    Keywords:  Drug acetylation; Drug glutathionylation; Drug metabolism; Drug oxidation; Drug sulfation; PharmMet DB
    DOI:  https://doi.org/10.1016/j.dmd.2025.100183
  28. Se Pu. 2025 Nov;43(11): 1209-1221
    [Magnetic one-step purification-high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry for the determination of 54 veterinary drug residues in carp].
    Yu-Peng Wen, Wen-di Huo, Chao-Ying Zhang, Huan Liu, Ying-Chun Mu, Li-Dong Wu, Jin-Cheng Li.
      Aquatic food products contain interfering substances， such as fats and proteins， which affect the analysis and detection of target compounds by significantly reducing detection efficiency and contaminating detection instruments. Effectively removing interfering substances is a key step in the field of food quality testing. To reduce the influence of interfering substances in aquatic products， this study established a detection method for 54 veterinary drug residues in carp by using magnetic one-step purification combined with high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry （HPLC-QTRAP-MS/MS）. The purification process uses solvothermally synthesized spherical magnetite as the magnetic material. This material is strongly magnetic and enables impurities to be efficiently separated and collected. Additionally， a metal-organic framework material with an amino-rich surface （ZIF-67） was self-assembled to serve as the purifying agent. ZIF-67 is particularly effective at removing polar impurities during the magnetic one-step purification process. Scanning electron microscopy confirmed the successful synthesis of both materials， ensuring their structural integrity and suitability for the intended application.Acetonitrile was chosen as the extraction solvent owing to its excellent solubilizing properties. Furthermore， formic acid was added to enhance the extraction efficiencies of acidic veterinary drugs. Anhydrous sodium sulfate was used to remove water from the matrix， thereby further improving the purity of the extract. The extract underwent magnetic one-step purification following centrifugation， which effectively removed interfering substances while preserving the target compounds. Chromatographic separation was achieved using the C18 RRHD column （15 cm×4.6 mm， 2.7 µm）， with 5 mmol/L ammonium acetate solution （containing 0.2% formic acid） and 0.2% formic acid in methanol used as mobile phases. HPLC-QTRAP-MS/MS was then used to separate and determine the analytes， with data collected in multiple reaction monitoring （MRM） mode. The 54 veterinary drugs were quantitatively analyzed using matrix-matched standard curves， thereby ensuring accurate quantification even for complex matrices. Various parameters that affect the purification fillers were investigated to optimize the purification process. Octadecyl-bonded silica gel （C18） was used as an auxiliary purification material as it is known to effectively remove non-polar impurities. Combining 60 mg of ZIF-67 with 50 mg of C18 yielded the best purification results. All 54 compounds exhibited weak matrix effects after pretreatment and purification， with a value of only 18.11% recorded for the compound that exhibited the most significant matrix effect. The 54 veterinary drugs exhibited excellent linear relationships between response and concentration in the 0.5-50.0 µg/L range， with all coefficients of determination （r²） above 0.99. Detection and quantification limits of 0.5-1.0 and 1.0-2.0 µg/kg were recorded， respectively. The performance of the method was evaluated by spiking blank carp matrix samples at three concentrations （2.0， 5.0， and 20.0 µg/kg）. The recovery rates of the spiked samples ranged between 81.34% and 109.85%， and intra-day and inter-day relative standard deviations were less than 10%. The rapidity and accuracy of the developed method were assessed through comparisons with existing veterinary-drug detection methods， which revealed that the developed method is more sensitive and repeatable. Furthermore， the feasibility of the established detection method was verified by testing commercial aquatic products. No target veterinary drugs were detected in actual samples， which highlights the reliability of the developed method. In conclusion， the method developed in this study provides an effective means of monitoring veterinary drugs in carp. It meets the requirements of economy， simplicity， and efficiency while exhibiting high sensitivity and reproducibility. The developed approach represents a significant advancement in the field of aquatic food safety testing and is expected to ensure more accurate and reliable detection of veterinary drug residues.
    Keywords:  crap; high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry （HPLC-QTRAP-MS/MS）; magnetic one-step purification; veterinary drugs
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.12010
  29. Rapid Commun Mass Spectrom. 2026 Feb 15. 40(3): e10169
    Direct Identification and Quantification of Flavonoids and Their Structural Isomers Using Ambient Ionization Tandem Mass Spectrometry.
    Yanqiu Wang, Liping Xu, Tiange Gu, Hongli Li, David Da Yong Chen.
       RATIONALE: Flavonoids are phenolic compounds with many health-benefiting properties. However, differentiating different types of flavonoids and their isomers is challenging due to their highly similar structures of various subtypes and different numbers and sites of substituents. Timely quality evaluation of flavonoid-based products is currently almost impossible.
    METHODS: An ambient ionization method of direct analysis in real time (DART) ion source and tandem mass spectrometry (MS) was used to characterize the fine structures of flavonoids. Different flavonoid subtypes and their isomers with varied numbers and sites of substituents were subjected to DART ionization and collision-induced fragmentation MS analysis.
    RESULTS: Seven classes of flavonoids, including methoxy-substituted compounds, exhibited distinctive fragmentation pathways such as retro-Diels-Alder reactions, cross-ring cleavages, and neutral losses. Many flavonoid isomers produced diagnostic MS2 and MS3 fragments through DART-tandem MS, enabling direct identification of various isomers within mixtures. An identification workflow was developed, culminating in the creation of a computational tool called FlavoFinder, which automatically determines flavonoid aglycone subtypes and their isomeric structures.
    CONCLUSIONS: The method and the structural elucidation program were successfully used for the qualitative and quantitative analysis of different flavonoid isomers from real samples. The analysis procedure is high-throughput and is capable of characterizing complex flavonoid structures without extensive sample pretreatment and front-end chromatographic separations.
    Keywords:  computer program; direct analysis in real time ionization; flavonoid aglycone subtype; flavonoid isomers; tandem mass spectrometry
    DOI:  https://doi.org/10.1002/rcm.10169
  30. J Integr Plant Biol. 2025 Nov 03.
    High-resolution dual-polarity molecular imaging platform unraveling spatial metabolic heterogeneity in multiple plant tissues.
    Guanhua Zhang, Chunxia Ma, Sheng Wang, Chuanzhi Kang, Chenglong Sun, Wei Liu, Chaogeng Lyu, Xiao Wang, Lanping Guo.
      Exploring the metabolic characteristics of different plant organs and tissues at a spatial level can help us to better understand the functional mechanisms of biological tissues and cells. Mass spectrometry imaging (MSI) provides a reliable tool for this purpose. However, its application for high-resolution metabolic mapping across various plant organs remains a significant challenge due to the intrinsic biological properties of plant samples and unfavorable analysis conditions. This study aimed to develop a novel MSI platform that can expand more diverse plant samples in spatial metabolomics research and enhance the detection efficiency of plant metabolites. The platform (AMG-LDI-MSI) based on an Au nanoparticles-loaded MoS2 and doped graphene oxide (Au@MoS2/GO) flexible film substrate combined with laser desorption/ionization (LDI)-MSI was established to enhance the detection and visualization of metabolites in various plant tissues. It has a non-sectioning, matrix-free, dual-ion mode imaging strategy, enabling high-throughput detection of metabolites and high-resolution molecular imaging within a micrometer scale. The Au@MoS2/GO as a new substrate can offer high sensitivity and molecular coverage for diverse plant metabolites (10 classes) under the positive and negative ion modes. Moreover, the AMG-LDI-MSI platform overcomes the limitations of plant tissues (e.g., fragile leaf, water-rich fruit, or lignified roots) for in situ imaging. We successfully applied the platform to map the metabolite spatial dynamics in different types of fresh tissues (rhizome, main root, branch root, fruit, leaf, and root nodule) from medicinal plants, obtained the high-quality mass spectral imaging data, and demonstrated the universality and applicability of the platform to multiple plant tissues. These results demonstrate the significant advantages of enhancing the detection of multiple tissue metabolites in plants and their high-resolution imaging. It has overcome the limitations of previously reported MSI methods, suggesting that it could become a widely used tool for deciphering metabolic networks in plant biology.
    Keywords:  flexible substrate; high‐resolution; mass spectrometry imaging (MSI); plant molecular imaging; tissue imaging
    DOI:  https://doi.org/10.1111/jipb.70061
  31. Microbiol Resour Announc. 2025 Nov 06. e0103025
    Targeted polar metabolomics data for Neurospora crassa wild type and mutant strains cultured on glucose.
    Alexander J Carillo, Lida Halilovic, Monique Quinn, Katherine A Borkovich.
      We present a metabolomics data set generated via high-performance liquid chromatography and high-resolution mass spectrometry for the filamentous fungus Neurospora crassa grown with glucose as the carbon source. A group of 121 polar primary metabolites was identified in wild type and gene replacement mutants lacking the heterotrimeric Gα subunits gna-1 or gna-3 or the non-receptor guanine nucleotide exchange factor ric8.
    Keywords:  filamentous fungi; high-pressure liquid chromatography; mass spectrometry; metabolomics; polar metabolites
    DOI:  https://doi.org/10.1128/mra.01030-25
  32. Shokuhin Eiseigaku Zasshi. 2025 ;66(4): 74-79
    [Development of a Qualitative Analytical Method for Milk, Egg, Buckwheat, and Peanut Allergens Using LC-MS/MS].
    Kana Kimoto, Ayano Hotta, Mayuko Oda, Mami Ogai, Maki Shimatani, Maki Kanda, Yuki Sadamasu, Takeo Sasamoto.
      LC-MS/MS was used to develop a qualitative analytical method for milk, egg, buckwheat, and peanut allergens. The measurement conditions and extraction and trypsin digestion process for protein pretreatment were optimized. A sample spiked with each allergen was analyzed to evaluate the performance of the analytical method, including the extraction process. Repeatability and intralaboratory precision met the target values for all allergens, confirming the stability of the analytical method. Furthermore, the S/N ratio, retention time, and peptide ratio demonstrated the usefulness of the method as a qualitative test. Moreover, for milk and egg allergens, the equivalence of the quantitative values calculated using the developed method to those of ELISA was confirmed; however, this equivalence could not be confirmed for commercially available processed foods and some quality control samples. For buckwheat and peanut allergens, the equivalence of the quantitative values to those of ELISA could not be confirmed in the spiked sample. These findings demonstrate that this analytical method is useful as a rapid and simple qualitative test. It is also an excellent alternative to ELISA as it produces less waste and reduces environmental burden.
    Keywords:  ELISA method; LC-MS/MS; buckwheat; egg; food allergen; milk; peanut
    DOI:  https://doi.org/10.3358/shokueishi.66.74
  33. J Chromatogr A. 2025 Nov 04. pii: S0021-9673(25)00860-X. [Epub ahead of print]1764 466516
    At-line analysis of antisense oligonucleotide purified fractions using fast chromatographic methods.
    Ahmed Elmekawy, Laura J Hayward, Masafumi Iwamoto, Jun Matsunami, Farzin Gharahdaghi.
      In the manufacturing of antisense oligonucleotides (ASOs), rapid analytical methods are crucial for efficient process control and quality assurance. However, current analytical techniques for ASO characterization are time-consuming, creating bottlenecks in the manufacturing process. To address this challenge, this study developed and implemented a fast chromatographic method for at-line analysis of ASO purified fractions, with a focus on integration with Multi-column Counter-current Solvent Gradient Purification (MCSGP). A fast 6.5-minute ion-pair liquid chromatography method with UV and MS detection was developed. Method parameters were optimised, including injection volume (2-4 µL) and sample concentration (0.1-0.2 mg/mL), to balance speed, sensitivity, and chromatographic performance. The method performance was comparable to a validated 40-minute LC-UV-MS method using various ASO samples, including conjugated crude and purified materials. The fast method demonstrated improved limits of detection and quantification compared to the conventional method, while maintaining linearity (R² > 0.99) and robust performance with high-salt MCSGP samples (up to 60 mM NaCl). Comparable accuracy was achieved for main components (UV purity, MS purity, % full length-n; %RSD < 2 %) across different sample types. The method's speed and compatibility with at-line and potential online analysis represent a significant advancement in ASO manufacturing process control.
    Keywords:  At-line analysis; Fast chromatographic method; Injection volume; MCSGP; Oligonucleotide; Sample concentration; Sample desalting
    DOI:  https://doi.org/10.1016/j.chroma.2025.466516
  34. Biomed Chromatogr. 2025 Dec;39(12): e70211
    Forced Degradation Studies of Dobutamine and Isolation, Identification, and Characterization of Potential Degradation Impurity by Preparative HPLC, LC-MS/MS. Development and Validation of Stability Indicative UPLC Method.
    Babu Reddy Goguladinne, Pulipaka Shyamala, K M V Narayana Rao.
      Dobutamine (DB) is a β1-adrenergic agonist with weak β2 activity and α1 selective activity, which is used clinically in cases of cardiogenic shock. Most of the available literature methods for DB that are LCMS incompatible have limitations for the identification of impurities. The current study describes forced degradation studies of DB and the identification of impurities by mass analysis, assigning structure to the major degradant, and the development and validation of an LCMS-compatible UPLC method. Impurity A (dopamine) highly polar impurity listed in the dopamine US Pharmacopeial Monograph, was retained in the developed method without an ion pair reagent. The method has been developed using Waters Acquity UPLC system equipped with Acquity UPLC HSS PFP column (2.1 mm × 100 mm, 1.8 μ), 0.05% TFA in water:ACN (95:5 %v/v) as Mobile Phase A, and 0.05% TFA in water:ACN (5:95 %v/v) as Mobile Phase B using a gradient program. DB degradation behavior was studied by subjecting it to acidic, basic, neutral, and oxidative conditions as per ICH guideline Q1A (R2). One of the major degradants was isolated by preparative HPLC and identified characterization by LCMS/MS and HRMS. The newly developed UPLC method was found to be specific with respect to degradation products, and the method has been validated according to the ICH guidelines.
    Keywords:  HRMS; LCMS; degradation products; dobutamine; dopamine; preparative HPLC
    DOI:  https://doi.org/10.1002/bmc.70211
  35. Food Chem. 2025 Oct 26. pii: S0308-8146(25)04077-4. [Epub ahead of print]496(Pt 3): 146825
    Novel LC-MS/MS strategies combining low-temperature partition extraction and DLLME for sensitive levamisole determination in fish.
    Lucas Victor Pereira de Freitas, Ana Luísa Bigliassi Alponti, Sarah Chagas Campanharo, Marina Alves Damaceno, Agnaldo Fernando Baldo da Silva, Marília Cristina de Oliveira Souza, Inácio Mateus Assane, Fabiana Pilarski, James Jacob Sasanya, Jonas Augusto Rizzato Paschoal.
      Sample preparation is a crucial and challenging step for determining veterinary drug residues (VDRs) in animal products. This study aimed to develop two novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of levamisole in fish fillet (muscle plus skin in natural proportion): Low-Temperature Partition Extraction (LTPE) and a combination of LTPE with Dispersive Liquid-Liquid Microextraction (DLLME). Optimal extraction conditions were established through multivariate optimization. Both methods were validated, demonstrating selectivity, precision (CV < 20 %), accuracy (recoveries 98 %-111 %), low matrix effects, and linearity in the ranges of 2-200 and 0.2-20 μg kg-1 for LTPE and LTPE-DLLME, respectively. The LTPE method was successfully applied in the analysis of real samples to evaluate levamisole residues in fish. To the best of our knowledge, this is the first study to combine LTPE and DLLME, representing a promising advance in sensitive and selective methods for routine monitoring of levamisole and potentially other VDRs in food-producing animals.
    Keywords:  Aquaculture; DLLME; Food safety; LC-MS/MS; LTPE; Veterinary drug residues
    DOI:  https://doi.org/10.1016/j.foodchem.2025.146825
This page is being maintained by Thomas Krichel. It was last updated on 2025‒11‒09 at 15:42.