bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–09–07
48 papers selected by
Sofia Costa, Matterworks
  1. Comparative evaluation of phenyl isothiocyanate derivatization and "dilute-and-shoot" methods for HPLC-MS/MS-based targeted metabolomics analysis of amine-containing metabolites in plasma samples.
  2. [Determination of N-（1，3-dimethylbutyl）-N'-phenyl-p-phenylenediamine-quinone in urine and dust by ultra performance liquid chromatography-tandem mass spectrometry].
  3. [Determination of strychnine in human serum by liquid chromatography tandem mass spectrometry].
  4. Knowledge and data-driven two-layer networking for accurate metabolite annotation in untargeted metabolomics.
  5. Simultaneous Bioanalytical Method Development and Validation of Navitoclax and Doxorubicin in Rat Plasma Using UHPLC-HESI-LTQ-MS.
  6. [Determination of 17 bisphenol compounds in human urine by solid supported liquid-liquid extraction-ultra-high performance liquid chromatography-tandem mass spectrometry].
  7. Local neighbor Normalization: Reconciling accurate normalization and heterogeneity recovery in large-scale metabolomics.
  8. [Determination of 26 perfluorinated and polyfluoroalkyl compounds in human serum by solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry].
  9. An automated Immunoaffinity liquid chromatography-tandem mass spectrometry assay for quantification of aldosterone in human plasma.
  10. A Method for Analysis of Free and Total Ropivacaine in Dog Plasma Using UHPLC-MS/MS.
  11. Open-Source System Suitability: Mass Spectrometry Query Language Lab (MassQLab).
  12. Suzetrigine in Equestrian Sports: Optimized Extraction and LC-HRMS Detection Strategies.
  13. Simulating Collision-Induced Dissociation Tandem Mass Spectrometry (CID-MS/MS) for the Blood Exposome Database Using Quantum Chemistry Methods - A Pilot Study.
  14. Development and validation of an LC-MS/MS method for measurement of porphobilinogen in urine.
  15. Mass spectrometry for clinical bioanalysis without chromatographic separation: bioequivalence for bupropion and its metabolites.
  16. Streamlined Sample Cleanup: Small Molecule Fractionation and Extraction Via Low-Volume Polymer Monolithic Columns for In-Line Analysis.
  17. Global metabolomics profiling of glucuronides in human plasma, fecal, and cerebrospinal fluid samples.
  18. Development and validation of a LC-MS/MS method for the detection of 38 benzodiazepines and 2 Z-drugs in blood.
  19. LC-MS/MS method for quantifying the HIV-1 broadly neutralizing antibody PGT 121.414.LS in human serum.
  20. [Fast determination of maduramicin ammonium in serum by high performance liquid chromatography-tandem mass spectrometry].
  21. [Rapid screening of 111 pesticides and veterinary drugs in livestock and poultry meat by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry].
  22. Using HILIC-MS/MS Method to Study Inositol Pyrophosphate.
  23. Multi-mycotoxin determination in human urine by UHPLC-MS/MS: An environmentally friendly and high-throughput approach.
  24. Unsupervised machine learning for mass spectrometry imaging data analysis with in vivo isotope labeling.
  25. Sustainable and renewable approaches in biomaterial-based integrated dried urine sampling/solid-phase extraction devices for drugs and metabolites: challenges, bioanalytical applications, and future directions.
  26. CLAW-MRM: Comprehensive Lipidomics Automation Workflow for Multiple Reaction Monitoring Using Large Language Models.
  27. [Simultaneous identification and detection of four novel isoxazoline drugs in bovine-origin foods using ultra-high performance liquid chromatography coupled to quadrupole/linear ion trap mass spectrometry].
  28. Development of a validated and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to simultaneous determination of crizotinib, alectinib and lorlatinib in human plasma.
  29. A Simple and Highly Sensitive Liquid Chromatographic-Mass Spectrometric Method for the Determination of Itraconazole and Its Major Metabolite in Human Plasma and Its Application to a Bioequivalence Study.
  30. The Power of Many: An Ensemble Approach to Spectral Similarity.
  31. Investigation of morantel metabolism and its application in veterinary drug residue screening.
  32. Overcoming bioanalytical challenges during the development of risdiplam for the treatment of spinal muscular atrophy.
  33. Rapid Dereplication of Bioactive Compounds in Plant and Food Extracts Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry.
  34. Large Language Model-Empowered Compound Collision Cross-Section Prediction.
  35. Automatic Identification of Potential Cellular Metabolites for Untargeted NMR Metabolomics.
  36. Miniaturized one-step extraction for the analysis of tetrahydrocannabinol ∆9- and ∆8- isomers, phytocannabinoids, and hexahydrocannabinol in whole blood using liquid chromatography tandem mass spectrometry.
  37. [Rapid determination of five antipyretic analgesics in surface water by online solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry].
  38. Capillary Electrophoresis Mass Spectrometry for Inositol (Pyro)phosphate Profiling.
  39. Profiling Multiple Steroid Subclasses Enabled by Rounded Turn SLIM Ion Mobility.
  40. Simultaneous Analysis of Cyproconazole and Tebuconazole in Suspension Concentrate Formulation by Reverse-Phase High-Performance Liquid Chromatography.
  41. Combining clinical chemistry with metabolomics for metabolic phenotyping at population levels.
  42. High-Specificity and Sensitivity Imaging of Neutral Lipids Using Salt-Enhanced MALDI TIMS.
  43. Development of a pseudotargeted metabolomics approach for relative quantification of free fatty acid double-bond isomers in beagle plasma.
  44. [Determination of 79 pesticide residues in fruits and vegetables by QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry].
  45. Analysis of Serum Serotonin by High-Performance Liquid Chromatography-Tandem Mass Spectrometry Using Online Solid-Phase Extraction.
  46. Development and application of a quantitative Liquid chromatography-tandem mass spectrometry method for multi-class organic contaminants in biological and environmental matrices using a desirability function-based optimization.
  47. Metabolite Fusion between Breath and Blood Enables More In-Depth Understanding of the Endogenous Metabolome.
  48. Unraveling the bridging region of Lenacapavir atropisomers by two-dimensional liquid chromatography.
  1. Anal Bioanal Chem. 2025 Aug 30.
    Comparative evaluation of phenyl isothiocyanate derivatization and "dilute-and-shoot" methods for HPLC-MS/MS-based targeted metabolomics analysis of amine-containing metabolites in plasma samples.
    Kangkang Xu, Markus Aigensberger, Franz Berthiller, Heidi E Schwartz-Zimmermann.
      Metabolomics, the study of small molecule metabolites in biological systems, is essential for disease diagnosis and biomarker discovery. A key consideration in developing targeted metabolomics methods using HPLC-MS/MS for human or animal plasma is whether to employ derivatization of amino acids, amino acid-related compounds, and biogenic amines. Derivatization with phenyl isothiocyanate (PITC) enhances ionization and LC-separation, but complicates sample preparation and introduces potential errors. This study focuses on (1) the validation of a PITC derivatization method employing reversed-phase (RP) HPLC-MS/MS analysis and (2) a comparison of two analytical approaches for targeted metabolomics analysis of animal and human plasma: the PITC derivatization-based RP-LC-MS/MS method and a "dilute-and-shoot" approach using hydrophilic interaction chromatography (HILIC)-MS/MS and RP-LC-MS/MS analysis. The derivatization method was validated for porcine plasma, assessing limits of detection, lower limits of quantification (LLOQs), linearity, repeatability, recovery, and trueness. Derivatization reduced LLOQs for derivatized compounds in pure solvent solutions but, due to higher dilution factors, resulted in similar LLOQs for derivatized compounds and higher LLOQs for non-derivatized compounds in plasma compared to the "dilute-and-shoot" method. Derivatization improved chromatographic separation of isomers and reduced carryover but introduced challenges such as matrix effects, coelution with impurities, and calibration issues. The "dilute-and-shoot" method performed better for non-derivatized compounds and was less error-prone. Both methods were applied to plasma from various species, demonstrating comparable concentrations for most metabolites. The results also emphasize the importance of using different approaches for cross-validation. Above all, this study highlights the strengths and limitations of both the derivatization method and the "dilute-and-shoot" approach, providing guidance for their application in targeted metabolomics.
    Keywords:  Amino acids; Biogenic amines; Derivatization; High-performance liquid chromatography; Mass spectrometry; Method comparison
    DOI:  https://doi.org/10.1007/s00216-025-06079-5
  2. Se Pu. 2025 Sep;43(9): 1025-1033
    [Determination of N-（1，3-dimethylbutyl）-N'-phenyl-p-phenylenediamine-quinone in urine and dust by ultra performance liquid chromatography-tandem mass spectrometry].
    Zhu-Liang-Zi Lu, Fen-Fang Deng, Zhi-Jun Bai, Rong-Fei Peng, Lei Tan.
      An ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method was established to determine N-（1，3-dimethylbutyl）-N'-phenyl-p-phenylenediamine-quinone （6PPD-Q） in human urine and dust in order to understand the internal and external exposure levels in humans. The sample preparation conditions were systematically investigated and the chromatographic conditions and MS parameters were optimized. Briefly， internal standard 13C6-6PPD-Q （0.1 ng） was added to a urine sample. Glutathione and NaCl were added after equilibration for 30 min. The mixture was then twice ultrasonically extracted with ethyl acetate. Internal standard 13C6-6PPD-Q （1.0 ng） was also added to a dust sample and the mixture was twice ultrasonically extracted with n-hexane. The combined organic phases were concentrated to near-dryness and then redissolved for instrumental determination， which was performed on a Phenomenex Kinetex F5 column （100 mm×3 mm， 2.6 μm）， with the target analyte gradient eluted using 10 mmol/L ammonium acetate aqueous solution containing 0.01% formic acid and acetonitrile. Positive electrospray ionization （ESI+） and multiple reaction monitoring （MRM） modes were used for identification purposes， and an isotope-labeled internal standard was added for quantification. Good linearities were achieved under the optimized conditions within the 0.01-4.00 and 0.01-20.0 μg/L ranges for urine and dust， respectively， with correlation coefficients of 0.999 9 and 0.999 3， respectively. Limits of detection （LODs） were 0.6 ng/L （urine） and 0.018 ng/g （dust）. Spiked recoveries of 6PPD-Q were 90.3%-94.1% at low， medium， and high spiked levels， with intra-day and inter-day precisions of 0.9%-5.9% and 1.1%-6.3%， respectively. Matrix-effect investigations revealed that 6PPD-Q exhibited weak matrix effects in urine and dust after correction with the isotopic internal standard. The developed method was used to analyze 120 human urine samples， which led to a 6PPD-Q detection frequency of 74.2%， with mass concentrations ranging from <LOD to 13 ng/L， with average and median mass concentrations of 2 and 1 ng/L， respectively. However， 6PPD-Q was detected with a frequency of 100% in 31 indoor dust samples with contents of 1.8-24.9 ng/g， and average and median contents of 5.23 -3.05 ng/g， respectively. The developed method is accurate， reliable， highly sensitive， and it is suitable for the rapid determination of 6PPD-Q in human urine and dust samples.
    Keywords:  N-（1，3-dimethylbutyl）-N′-phenyl-p-phenylenediamine-quinone （6PPD-Q）; dust; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）; urine
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.02010
  3. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2025 Aug 20. 43(8): 615-618
    [Determination of strychnine in human serum by liquid chromatography tandem mass spectrometry].
    X H Chen, C Qin, J Zhou.
      Objective: To establish a method for the determination of strychnine in human serum by liquid chromatography tandem mass spectrometry. Methods: In April 2024, the serum samples were extracted using alkaline ethyl acetate. Separation was achieved using a Waters HSS T3 column (100 mm×2.1 mm, 1.8 μm) with a linear gradient elution program of water/acetonitrile (containing 5 mmol/L ammonium formate and 0.1% formic acid) as the mobile phase. The analyte was detected by using electrospray ionization (ESI) in the positive multiple reaction monitoring (MRM) mode and quantified using an internal standard method. Results: A good linearity was obtained in the concentration range of 0.01~20.0 μg/L for target compound with the correlation coefficients≥0.999. The limitof detection (LOD) and the limit of quantitation (LOQ) were 0.003 μg/L and 0.01 μg/L, respectively. The average spiked recovery was 101.3%, the intra-day precision was 2.8%-5.4%, and the inter-day precision was 3.9%-6.3%, respectively. Conclusion: Liquid chromatogrphy tandem mass spectrometry method exhibits high sensitivity, strong specificity, and good accuracy, and can be used for the determination of strychnine in Homo sapiens serum.
    Keywords:  Chromatography, liquid; Serum; Strychnine; Tandem mass spectrometry
    DOI:  https://doi.org/10.3760/cma.j.cn121094-20240411-00160
  4. Nat Commun. 2025 Aug 30. 16(1): 8118
    Knowledge and data-driven two-layer networking for accurate metabolite annotation in untargeted metabolomics.
    Haosong Zhang, Xinhao Zeng, Yandong Yin, Zheng-Jiang Zhu.
      Metabolite annotation in untargeted metabolomics remains challenging due to the vast structural diversity of metabolites. Network-based approaches have emerged as powerful strategies, particularly for annotating metabolites lacking chemical standards. Here, we develop a two-layer interactive networking topology that integrates data-driven and knowledge-driven networks to enhance metabolite annotation. A comprehensive metabolic reaction network is curated using graph neural network-based prediction of reaction relationships, enhancing both coverage and network connectivity. Experimental data are pre-mapped onto this network via sequential MS1 matching, reaction relationship mapping, and MS2 similarity constraints. The generated networking topology enables interactive annotation propagation with over 10-fold improved computational efficiency. In common biological samples, it annotates over 1600 seed metabolites with chemical standards and >12,000 putatively annotated metabolites through network-based propagation. Notably, two previously uncharacterized endogenous metabolites absent from human metabolome databases have been discovered. Overall, this strategy significantly improves the coverage, accuracy, and efficiency of metabolite annotation and is freely available as MetDNA3.
    DOI:  https://doi.org/10.1038/s41467-025-63536-6
  5. J Chromatogr Sci. 2025 Aug 30. pii: bmaf046. [Epub ahead of print]63(8):
    Simultaneous Bioanalytical Method Development and Validation of Navitoclax and Doxorubicin in Rat Plasma Using UHPLC-HESI-LTQ-MS.
    Nitesh Rai, Deepak Kumar, Parul Sharma, Pirangi Srikanth, Shaik Khaja Moinuddin, Pramod Kumar, Sukhendu Nandi.
      A simple, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was successfully developed and validated to determine navitoclax and doxorubicin in rat plasma. Ketoconazole and daunorubicin were employed as internal standards to ensure accurate quantification and method consistency. The sample preparation involved a straightforward protein precipitation technique, which facilitated efficient extraction of the analytes from the plasma matrix. The resulting supernatant was subjected to drying using a vacuum concentrator and later reconstituted before being injected into the LC-MS system. Separation was carried out using a SHIMADZU Shim-pack GIST C-18 column with mobile phase, consisting of a carefully balanced mixture of acetonitrile and water with 0.1% formic acid, with a flow rate of 0.5 mL/min. This composition ensured efficient elution and minimized matrix effects, contributing to the method's robustness and reproducibility. The developed method was comprehensively validated according to regulatory guidelines, assessing various parameters including specificity, selectivity, sensitivity, calibration curve performance, precision, accuracy, stability and dilution integrity. This bioanalytical method has the potential to be extended to various clinical settings, enabling the monitoring drug metabolism, monitoring potential drug-drug interactions and understanding the pharmacokinetic profile and adverse effect potential of the combination therapy.
    DOI:  https://doi.org/10.1093/chromsci/bmaf046
  6. Se Pu. 2025 Sep;43(9): 1014-1024
    [Determination of 17 bisphenol compounds in human urine by solid supported liquid-liquid extraction-ultra-high performance liquid chromatography-tandem mass spectrometry].
    Yu'e Jin, Lan-Ge Zhang, Jing-Xian Zhou, Jin-Jing Ma, Li-Li Yuan, Ping Xiao, Guo-Quan Wang.
      Bisphenol A （BPA） and its analogs are collectively termed bisphenol compounds （BPs）， which are predominantly utilized in the manufacturing of polycarbonate plastics and epoxy resins. BPs are ubiquitous in diverse environmental matrices， human tissues， and metabolic products. Extensive research has demonstrated that BPs exert adverse effects on the nervous， reproductive， immune， and metabolic systems. After exposure in humans， BPs are primarily excreted in urine. Consequently， the development of efficient and robust analytical methods for BPs quantification in urine is essential for assessing population exposure levels. In this study， solid supported liquid-liquid extraction （SLE） was combined with ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） technology to establish a high-throughput determination method for 17 BPs in human urine. After enzymatic hydrolysis treatment of the samples， the steps of fully automatic SLE purification， nitrogen blowing concentration and redissolution were carried out successively. Separation was performed using a CAPCELL PAK ADME chromatography column （100 mm×2.1 mm， 2 μm）， and gradient elution was carried out with 0.05 mmol/L ammonium fluoride aqueous solution and 0.05 mmol/L ammonium fluoride methanol solution as the mobile phases. MS detection was carried out using the electrospray ionization （ESI） negative ion scanning mode under the multi-reaction monitoring （MRM） mode. Qualitative analysis was conducted based on retention time and ion abundance ratio， and quantitative analysis was performed using the internal standard method. Under the optimized conditions， 17 BPs can be effectively separated. The linear relationships of the 17 BPs within the corresponding mass concentration ranges were good， and the correlation coefficients （r） were ≥0.998 6， the limits of detection （LODs） and quantification （LOQ） were 0.002-0.489 μg/L and 0.005-0.986 μg/L， respectively. Children's mixed urine samples with low background content were selected as the matrix， and then spiked recovery tests were conducted at three spiked levels （low， medium and high）. The results showed that the recoveries of 17 BPs were 61.1%-121.7%， the intra-day RSDs were 1.3%-11.2%， and the inter-day RSDs were 3.7%-19.0%. This method was used to determine 50 random urine samples， and the results showed that a total of 11 BPs were detected. Among them， bisphenol S （BPS） and BPA had the highest detection rates， which were 98.0% and 86.0% respectively， and the median detection levels were 0.075 μg/L and 0.829 μg/L respectively. This method is simple to operate， sensitive and reliable. It is suitable for the rapid quantitative analysis of 17 BPs in human urine and can provide effective technical support for the risk assessment of BPs exposure in the population.
    Keywords:  bisphenol compounds （BPs）; human urine; solid supported liquid-liquid extraction （SLE）; ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.11032
  7. Anal Chim Acta. 2025 Oct 22. pii: S0003-2670(25)00834-7. [Epub ahead of print]1372 344440
    Local neighbor Normalization: Reconciling accurate normalization and heterogeneity recovery in large-scale metabolomics.
    Keyi Lu, Yaru Liu, Kian-Kai Cheng, Fanjing Guo, Lingli Deng, Jiyang Dong.
       BACKGROUND: Metabolomics studies often grapple with the dilution effect, where sample concentrations vary due to inconsistent handling or biological diversity, particularly in samples like urine, saliva, or cell extracts. This variation can mask true metabolic differences, complicating data interpretation. Traditional normalization methods, such as Constant Sum Normalization (CSN), Probabilistic Quotient Normalization (PQN), and Maximal Density Fold Change (MDFC), assume that all samples share a certain invariant statistic and overlook data heterogeneity, potentially erasing the dataset's heterogeneity essential for distinguishing biological subgroups.
    RESULTS: To address this, we introduce Local Neighbor Normalization (LNN), a novel approach that corrects for dilution effects while preserving the intrinsic variability of metabolomics data. LNN identifies a neighbor set for each sample based on similarity metrics and normalizes each sample against a tailored reference spectrum derived from these neighbors. Through comprehensive evaluations on both simulated and real metabolomics datasets from NMR, GC-MS, and LC-MS platforms, LNN demonstrated superior performance over CSN, PQN, and MDFC. Specifically, it achieved better elimination of dilution effects, recovery of inter-sample heterogeneity and inter-metabolite correlations, as evidenced by metrics such as the D-statistic and correlation recovery rates. Notably, LNN excels in datasets with over 50 % differential metabolites, safeguarding local data structures critically for downstream analyses like biomarker discovery.
    SIGNIFICANCE AND NOVELTY: LNN constructs sample-specific reference spectra based on a local neighbor set. This approach ensures that normalization accounts for dilution effects without compromising local structure of the data, which is crucial for biological interpretation. Additionally, LNN demonstrates superior performance in recovering inter-sample heterogeneity and metabolite correlations, especially in datasets with high proportions of differential metabolites. This method's versatility, robustness against noise, and applicability across various metabolomics platforms make it a significant advancement in the field.
    Keywords:  Dilution effect; Heterogeneity; Local neighbor normalization; Metabolomics
    DOI:  https://doi.org/10.1016/j.aca.2025.344440
  8. Se Pu. 2025 Sep;43(9): 1005-1013
    [Determination of 26 perfluorinated and polyfluoroalkyl compounds in human serum by solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry].
    Ying-Xiao Yue, Ya-Ting Bian, Yu-Fan Cheng, Lu He, Dan Wang, Pei-Xia Yan, Wei Yan, Gui-Ying Liu, Huan Song, Liang-Po Liu.
      Perfluorinated and polyfluoroalkyl compounds （PFASs） represent a category of synthetic chemicals renowned for their environmental persistence. Owing to their hydrophobic， oleophobic， and high-temperature-resistant properties， PFASs are extensively utilized in industrial， agricultural， and civilian sectors， including applications in leather， textiles， flame-retardant materials， lubricants， and coatings， among others. PFASs can accumulate within the human body， exhibiting multi-organ toxicity. Continuous monitoring of PFASs with ambiguous toxicity profiles is vital for evaluating human exposure and associated health risks. Consequently， the establishment of a high-throughput and highly sensitive detection method is of paramount importance for accurately assessing the exposure levels of PFASs in the human body. In this study， a commercial high-throughput HMR-Lipid 96-well solid-phase extraction plate was adopted， combined with high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）， to establish a simple， efficient method that can simultaneously quantitatively detect 26 PFASs in human serum. Serum samples were extracted using the HMR-lipid 96-well solid-phase extraction plate. The Phenomenex C18 chromatography column （250 mm×4.6 mm， 5 μm） was used as the capture column and connected between the liquid chromatography mixer and the autosampler to avoid high background pollution. The target compounds were separated by the Accucore C18 chromatography column （100 mm×2.4 mm， 2.6 μm） and analyzed using the electrospray ionization with negative ion scanning mode and multiple reaction monitoring （MRM） mode. The methodological validation results indicated that the 26 PFASs had good linear relationships within the range of 0.2-100 ng/mL， with correlation coefficients （r） of 0.995 1-0.999 9. The limits of detection （LODs） and quantification （LOQs） were 0.01-0.15 ng/mL and 0.02-0.47 ng/mL， respectively. At three spiked levels of low， medium and high， the recoveries of the 26 PFASs ranged from 80.1% to 119.5%， and the relative standard deviations （RSDs） ranged from 0.5% to 11.9%. This method has the advantages of high sensitivity， good accuracy， simple operation， fast extraction speed， low reagent consumption and small sample volume required. It is suitable for large-scale population biological monitoring and provides a scientific method support for accurately assessing the exposure of PFASs in the human body and its potential health risks.
    Keywords:  HMR-lipid 96-well solid-phase extraction plate; high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）; human serum; perfluorinated and polyfluoroalkyl compounds （PFASs）
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.10002
  9. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 23. pii: S1570-0232(25)00327-7. [Epub ahead of print]1266 124773
    An automated Immunoaffinity liquid chromatography-tandem mass spectrometry assay for quantification of aldosterone in human plasma.
    Liang-Xing Li, Jian-You Xue, Wan-Ying Lin, Xiao-Feng Yin, Ting-Ting Luo, Zhi-Liang Cai, Chao-Chao Wu, Qiang Gao, Xin Li, Ru-Yi Zhang.
      The diagnosis of primary aldosteronism (PA) relies on the accurate determination of aldosterone. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has long been considered the gold standard for aldosterone quantification but it is hindered by labor-intensive sample preparation. To address this, we developed an immunoaffinity-mass spectrometry (iMS) assay on a fully automated device combining anti-aldosterone antibodies with stable isotope-labeled internal standards (IS). This method completes sample preparation within 15 min for at least six parallel samples in parallel with minimal manual intervention. The key performance metrics include a lower limit of quantitation (LOQ) of 50 pg/mL, recovery rates between 105.1 and 113.9 %, and linearity in the range of 50-2000 pg/mL (R2 = 0.9993). Inter-assay coefficient of variation (CV) ranged from 2.33 % to 3.91 %. In addition, plasma aldosterone concentrations by iMS and immunoassay had a high correlation coefficient (R = 0.947). Overall, this automated high-throughput platform delivers clinical-grade sensitivity, precision, and scalability, making it suitable for routine testing and adaptable for other clinical analytes.
    Keywords:  Aldosterone; Automated immunoaffinity; LC-MS/MS; Primary aldosteronism
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124773
  10. Biomed Chromatogr. 2025 Oct;39(10): e70214
    A Method for Analysis of Free and Total Ropivacaine in Dog Plasma Using UHPLC-MS/MS.
    Natali Verdier, Karin Hummel, Ebrahim Razzazi-Fazeli.
      Ropivacaine is a local anesthetic commonly used in veterinary anesthesia. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantify free and total ropivacaine in dog plasma, which included rapid equilibrium dialysis. The method was validated for selectivity, specificity, matrix effect, calibration curve and range, accuracy and precision, carry-over, stability, and reinjection reproducibility according to the International Conference on Harmonization M10 guidelines. After ultra-high performance liquid chromatographic (UHPLC) separation, detection and quantification of ropivacaine was performed using a triple quadrupole tandem mass spectrometer with electrospray ionization. LC-MS method validation was carried out in a range of 0.05-1000 ng/mL ropivacaine in dog plasma in two dilutions (1:1 and 1:4). The precision and accuracy of the method were determined at four concentration levels and ranged from 0.40% to 5.30% and 85.50% to 113.30%, respectively. The lower limit of quantification was as low as 0.30 and 0.05 ng/mL, for the quantitation of protein-bound (1:4) and free (1:1) ropivacaine, respectively. All validation parameters met acceptance criteria. This UHPLC-MS/MS method was successfully applied in a clinical study that involved the intraperitoneal instillation of ropivacaine to anesthetized dogs and can be used to quantify free and total ropivacaine in dog plasma.
    Keywords:  liquid chromatography; mass spectrometry; method validation; rapid equilibrium dialysis; ropivacaine
    DOI:  https://doi.org/10.1002/bmc.70214
  11. Rapid Commun Mass Spectrom. 2025 Dec 15. 39(23): e10132
    Open-Source System Suitability: Mass Spectrometry Query Language Lab (MassQLab).
    Heather L Winter, Dylan Johnson, Alan K Jarmusch.
       RATIONALE: Reproducible analytical instrumentation system performance is critical for mass spectrometry, particularly metabolomics, aptly named system suitability testing. We identified a need based on literature reports that stated only 2% of papers performed system suitability testing.
    METHODS: We report MassQLab, built upon open-source, vendor-agnostic software called the mass spectrometry query language (MassQL). MassQL, implemented in MassQLab, provides freedom for researchers to choose their analyte/s, mass spectrometry system (including liquid chromatography-mass spectrometry), and metrics of performance.
    RESULTS: In this report, we describe the use of MassQLab, demonstrate the construction of the required MassQL query, common metrics of performance (i.e., extracted ion chromatograms), uncommon metrics (i.e., MS/MS product ion spectra), and discuss insights gained about performance-including issues requiring correction prior to sample analysis.
    CONCLUSIONS: MassQLab is a flexible solution for system suitability testing for mass spectrometry-based analytical measurements. Deficits in analytical performance, while unavoidable and rare, were noted prior to data collection and corrected. The open-source and adaptable nature of MassQLab will empower researchers and lead to improved implementation of system suitability testing.
    Keywords:  computational mass spectrometry; data science; lipidomics; metabolomics; quality assessment; quality control
    DOI:  https://doi.org/10.1002/rcm.10132
  12. Rapid Commun Mass Spectrom. 2025 Dec 15. 39(23): e10135
    Suzetrigine in Equestrian Sports: Optimized Extraction and LC-HRMS Detection Strategies.
    Meleparappil Muhammed Ajeebsanu, Shino Ann Koshy, Abdul Khader Karakka Kal, Michael Benedict Subhahar, Tajudheen K Karatt, Moses Philip.
       RATIONALE: Suzetrigine, a recently approved NaV1.8 sodium channel blocker, shows strong potential in the treatment of neurological, psychiatric, and pain-related conditions. Its peripheral selectivity enables effective pain management while avoiding central nervous system complications and addiction risks linked to opioid use. Following FDA approval in January 2025, concerns have emerged regarding its possible misuse for performance enhancement in sports, highlighting the need for reliable detection tools in doping control.
    METHODS: An analytical procedure was designed and validated to detect suzetrigine in equine urine and plasma. Different chromatographic columns, mobile phase compositions, and ionization modes were systematically tested. Extraction efficiency was evaluated using solid-phase extraction (SPE), liquid-liquid extraction (LLE), and dilute-and-inject techniques to identify the most suitable approach for sensitivity and recovery.
    RESULTS: Of the various chromatographic columns evaluated, the AQUA C18 column (3.0 μm, 4.6 × 150 mm) exhibited the best separation performance. Among the extraction techniques tested, LLE optimized at specific pH levels and solvent conditions consistently achieved superior recovery rates and lower limits of detection. The fully validated procedure, utilizing high-resolution mass spectrometry, demonstrated excellent sensitivity, reproducibility, and robustness, making it suitable for routine detection of suzetrigine in biological matrices.
    CONCLUSIONS: The validated approach offers a reliable tool for doping control laboratories to detect suzetrigine in equestrian samples. Beyond equine testing, this protocol provides a methodological framework that can be extended to broader anti-doping programs, supporting the monitoring of emerging substances with misuse potential in sports.
    Keywords:  LC‐HRMS analysis; NaV1.8 sodium channel modulator; anti‐doping detection; sample extraction methods; suzetrigine
    DOI:  https://doi.org/10.1002/rcm.10135
  13. J Am Soc Mass Spectrom. 2025 Aug 28.
    Simulating Collision-Induced Dissociation Tandem Mass Spectrometry (CID-MS/MS) for the Blood Exposome Database Using Quantum Chemistry Methods - A Pilot Study.
    Jesi Lee, Dinesh Barupal.
      A significant number of compounds in exposome databases and chemical inventories lack mass spectral data due to the nonavailability of reference standards. To address this limitation, computational chemistry methods can be utilized to extend mass spectral libraries for a set of chemicals. In this pilot study, we employed quantum-chemistry-based software QCxMS to generate collision-induced dissociation mass spectra for 121 compounds from the Blood Exposome Database. We developed a scalable computational framework that integrates QCxMS and additional tools, utilizing a grid-based parameter selection strategy and defined coverage criteria. Our approach systematically explored protomeric isomers and applied predefined parameter combination sets sequentially based on molecular structures. This workflow produced high-quality in silico spectra for 81 compounds that achieved entropy similarity scores ≥700 and at least two matching fragment ions against the NIST23 library, yielding 71% spectral coverage. These results highlight the importance of optimizing simulation parameters and accounting for protomeric diversity to enhance the spectral quality and computational efficiency. This workflow provides a practical strategy to add mass spectral data for most compounds in the Blood Exposome Database at reasonable computational cost, supporting the spectral library expansion for improved compound annotation in exposomics.
    Keywords:  Blood Exposome; Mass Spectra Simulation; Metabolomics; QCCIDMS; QCxMS; Semiempirical methods
    DOI:  https://doi.org/10.1021/jasms.5c00179
  14. Clin Chim Acta. 2025 Aug 28. pii: S0009-8981(25)00458-9. [Epub ahead of print] 120579
    Development and validation of an LC-MS/MS method for measurement of porphobilinogen in urine.
    Mark M Kushnir, Laura Sinden, Elizabeth L Frank.
       INTRODUCTION: Measurement of urinary porphobilinogen (PBG) is used to diagnose acute porphyria, which results from disorders of heme biosynthesis. In response to clinical demand and to provide optimal patient care, we developed a simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for measurement of PBG in urine.
    MATERIALS AND METHODS: Aliquots (50 µL) of urine were spiked with a stable isotope-labeled internal standard (13C2,15N-PBG); PBG was extracted using anion exchange solid phase extraction, and the extracts were analyzed using reverse phase chromatographic separation with mass spectrometry detection. Injection-to-injection time was 5.5 min. Measured concentrations were normalized to creatinine (Cr) concentration in random specimens; PBG excretion per day was calculated for 24-h urine collections.
    RESULTS: Mean recovery was 99.7 %; total imprecision of analysis was < 16 %. Limit of quantification of the method was 0.1 µmol/L and the upper limit of linearity was 100 µmol/L. Reference intervals (RI) for the ratio of PBG to Cr concentration in urine samples from apparently healthy adults were < 0.19 and < 0.17 mmol PBG/mol Cr for women and men, respectively. The RI for 24-h excretion established using LC-MS/MS was 0.39-1.40 µmol/24-h. Percent positivity determined following implementation of the LC-MS/MS method (7.6 %) compared well with the positivity rate of the spectrophotometric method (7.7 %).
    CONCLUSIONS: This LC-MS/MS method has sufficient specificity and sensitivity for analysis of urinary PBG in pathologic samples and samples from healthy subjects. RIs for PBG in random and 24-h urine collections were established and the clinical performance of the assay was evaluated.
    Keywords:  Acute hepatic porphyria; Liquid chromatography; Mass spectrometry; Porphobilinogen; Reference intervals
    DOI:  https://doi.org/10.1016/j.cca.2025.120579
  15. Bioanalysis. 2025 Sep 05. 1-11
    Mass spectrometry for clinical bioanalysis without chromatographic separation: bioequivalence for bupropion and its metabolites.
    Jinhui Zhang, Patrick J Faustino.
       BACKGROUND: High-throughput solid-phase extraction coupled with tandem mass spectrometry (HT-SPE-MS/MS) is an automated sample delivery system to mass spectrometry that operates without chromatographic separation. The typical analysis time per sample using this platform is 10-30 s. While the HT-SPE-MS/MS system has demonstrated efficacy for in vitro assays, its application to the analysis of biological samples from in vivo bioavailability and bioequivalence studies presents challenges due to the complexity of the sample matrix. Three critical issues - matrix effect, specificity, and carryover - have not been thoroughly evaluated in complex biological matrices such as plasma.
    RESEARCH DESIGN AND METHODS: This study assessed the feasibility of utilizing HT-SPE-MS/MS for the analysis of three metabolically related compounds (bupropion, hydroxybupropion, and threobupropion) in human plasma samples from a clinical bioequivalence study. Critical bioanalytical parameters, including matrix effect, specificity, accuracy, precision, and carryover, were systematically investigated.
    RESULTS: These methods were subsequently applied to a bioequivalence study of bupropion. The HT-SPE-MS/MS approach achieved comparable accuracy, precision, linearity, and sensitivity to conventional ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) methods, while offering 20- to 30-fold higher analysis speeds.
    CONCLUSION: The results of this study indicate that the HT-SPE-MS/MS system shows potential for high-throughput in vivo bioanalysis, particularly in bioavailability and bioequivalence studies.
    Keywords:  Mass spectrometry; SPE-MS/MS; bioequivalence; bupropion; matrix effect
    DOI:  https://doi.org/10.1080/17576180.2025.2557187
  16. J Chromatogr Sci. 2025 Aug 30. pii: bmaf049. [Epub ahead of print]63(8):
    Streamlined Sample Cleanup: Small Molecule Fractionation and Extraction Via Low-Volume Polymer Monolithic Columns for In-Line Analysis.
    Ischa Bremer, Charles Clark, Bert Wouters, Amy Harms, Thomas Hankemeier.
      Polymer monoliths are stationary-phase materials for liquid chromatography and solid-phase extraction. Their porous structure, tuneability and simple synthesis enable tailoring to specific analysis requirements in analytical chemistry. Typically, polymer monoliths are used to separate larger biomolecules. Due to their lower binding capacity, the applications of polymer monoliths for the chromatographic separation of small molecules remain limited. However, recent literature has shown that polymer monoliths have the potential for the extraction of small molecules. In this research, butyl methacrylate-co-ethylene glycol dimethacrylate polymer monoliths were synthesized using localized UV polymerization in capillaries. The performance of reversed-phase polymer monoliths in automated in-line solid-phase extraction-mass spectrometry was demonstrated by the analysis of endocannabinoids from neat standard mixes and spiked cell culture media without prior sample preparation. The synthesized monoliths exhibited a binding capacity of 1896 pmol. Furthermore, we showed the repeatability of the monolith synthesis, with a variance in permeability of 19%. The system's stability is demonstrated through the analysis of multiple batches, comparing different monoliths and reusing the same monolith repeatedly, resulting in relative standard deviations (RSDs) below 20% for all extracted compounds. This automated method with hyphenated mass spectrometry improves throughput over previous manual monolithic extractions for small molecules.
    DOI:  https://doi.org/10.1093/chromsci/bmaf049
  17. Anal Bioanal Chem. 2025 Sep 01.
    Global metabolomics profiling of glucuronides in human plasma, fecal, and cerebrospinal fluid samples.
    Ioanna Tsiara, Mario S P Correia, Fan Yang, Weiming Zeng, Pauline Seeburger, Belén Hervás Povo, Iben Lundgaard, Manuel Menéndez-González, Miroslav Vujasinovic, J-Matthias Löhr, Daniel Globisch.
      Glucuronidation is the major phase II biotransformation reaction that facilitates the clearance of exogenous compounds from the human body. Glucuronidated metabolites have been investigated in urine samples at a broad scale; however, their characterization in other human biospecimens is underexplored. Our study has now performed a comprehensive profiling of glucuronides in plasma, fecal, and cerebrospinal fluid (CSF) of humans. We performed a mass spectrometry-based analysis that combines enzymatic hydrolysis with a β-glucuronidase to selectively cleave the glucuronic acid moiety, a developed in-house glucuronide identification pipeline, and enzymatic synthesis of standard metabolites. In total, we identified 32 glucuronidated metabolites across the three sample types in both negative and positive mass spectrometry ionization modes using advanced MS/MS fragmentation analysis. We have utilized a straightforward enzymatic synthesis of glucuronidated metabolites for annotation at the highest confidence level. Among the identified conjugates, we detected glucuronides of different compound classes including drugs, bile acid derivatives, steroid conjugates, and phenolic compounds. Unexpectedly, we validated the glucuronides of acetaminophen and propofol in CSF samples, the latter representing a novel observation that highlights the importance of investigating phase II metabolites in uncommon sample types.
    Keywords:  Enzymatic synthesis; Glucuronides; Gut microbiota; Metabolomics; Phase II modifications
    DOI:  https://doi.org/10.1007/s00216-025-06082-w
  18. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 26. pii: S1570-0232(25)00326-5. [Epub ahead of print]1266 124772
    Development and validation of a LC-MS/MS method for the detection of 38 benzodiazepines and 2 Z-drugs in blood.
    Yu Liu, Xin-Ze Liu, Zhen-Shuo Guo, Ping Xiang, Hui Yan.
      Benzodiazepines and Z-drugs are commonly used as prescription medications to treat anxiety, epilepsy, insomnia, and alcohol withdrawal syndrome, but their use can lead to tolerance, dependence, and withdrawal reactions if taken against official guidelines. Furthermore, designer benzodiazepines, most of which lack clinical and toxicological data, have entered the illicit drug market as new psychoactive substances and are used for recreational purposes. Their abuse can cause confusion, memory loss, respiratory depression, and even death, especially when combined with other sedative-hypnotics or alcohol. Therefore, new qualitative and quantitative methods for benzodiazepines and Z-drugs are needed for use in forensic and clinical toxicology. This study explored the sample preparation and liquid-liquid extraction using 0.5 mL blood samples and 2 mL of extraction solvent for analysis of these drugs by liquid chromatography-tandem mass spectrometry and multiple reaction monitoring. The benzodiazepines were separated on a pentafluorophenylpropyl (PFPP) column using a mobile phase gradient consisting of A (water, 0.1 % formic acid, 5 % acetonitrile, and 20 mmol/L ammonium acetate) and B (acetonitrile) for 9 min. Validation steps confirmed that the method demonstrated good selectivity, sensitivity (limit of detection: 0.2 ng/mL, lower limit of quantification: 0.5 ng/mL), linearity (R2 ≥ 0.99), accuracy, and precision (<20 %). Matrix effects ranged from 35 to 126 %, and recoveries ranged from 17 to 99 %, with 35 compounds having recoveries of more than 50 %. The method was successfully applied for the identification and quantification of benzodiazepines and nonbenzodiazepines in blood samples from 15 authentic poisoning cases. Ten analytes were detected: alprazolam (130.2-575.3 ng/mL), hydroxyalprazolam (2.3-37.3 ng/mL), clonazepam (11.7-773.2 ng/mL), lorazepam (63.4-166.9 ng/mL), 7-aminoclonazepam (17.4-385.3 ng/mL), oxazepam (2.6-964.1 ng/mL), diazepam (227.2 ng/mL), nordazepam(22.4 ng/mL), zolpidem (11.8-64.0 ng/mL), midazolam (70.2 ng/mL), and hydroxymidazolam (162.8 ng/mL).
    Keywords:  Benzodiazepines; Human poisoning cases; LC-MS/MS; Z-drugs
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124772
  19. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 27. pii: S1570-0232(25)00328-9. [Epub ahead of print]1266 124774
    LC-MS/MS method for quantifying the HIV-1 broadly neutralizing antibody PGT 121.414.LS in human serum.
    Connor E Gould, Qing Ma, Raymond Cha, Kevin J Zemaitis, Robin DiFrancesco, Gene D Morse, Troy D Wood.
      Quantitation of human immunodeficiency virus-1 (HIV-1) broadly neutralizing antibodies (bNAbs) in human serum is required for clinical trials investigating the pharmacokinetics, pharmacodynamics, and drug interactions of these treatments. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is gaining interest as an alternative to ligand binding for therapeutic antibody quantitation in serum. We report the validation of a method using nonspecific purification and targeted LC-MS/MS to quantify PGT 121.414.LS (a bNAb in development for HIV-1 prevention and treatment) in human serum. High-resolution spectra of tryptic peptides derived from the variable region were obtained on an Orbitrap for surrogate peptide selection, followed by multiple reaction monitoring using triple quadrupole mass spectrometry. Surrogate peptides were evaluated for linearity and reproducibility across the therapeutic concentration range using immunopurification or ammonium sulfate precipitation. Using ammonium sulfate precipitation, linear calibration curves were validated over 10-500 μg/mL (LLOQ at 10 μg/mL) using stable isotope labeled peptide internal standards. Method accuracy and reproducibility were evaluated using quality control samples (QCs) at four concentrations in the linear range. The average concentrations of all QCs fell within ICH M10 acceptance criteria. Matrix effects were investigated at the low and high QC concentrations across six lots of human serum. Dilutional integrity, stability, and effects of hemolysis were also assessed. The method exhibits minimal carryover and negligible crosstalk. The assay provides accurate quantification of PGT 121.414.LS in serum over the range of concentrations anticipated in specimens from treated persons living with HIV (PLWH) after initial dosing and prior to subsequent dosing of PGT 121.414.LS.
    Keywords:  Antibodies; HIV-1; Immunopurification; LC-MS/MS; Proteomics; Quantitation; Surrogate peptide
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124774
  20. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2025 Aug 20. 43(8): 619-622
    [Fast determination of maduramicin ammonium in serum by high performance liquid chromatography-tandem mass spectrometry].
    B Zhou, B W Cheng, J Ma, F Dong, R C Liu, H L Li.
      Objective: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the fast determination of maduramicin ammonium (MAD) in rat serum. Methods: In February 2024, rat serum samples were selected and directly injected after extraction and purification with methanol: acetonitrile (1: 1), separated on a C18 chromatographic column, and gradient-eluted using 0.1% formic acid aqueous solution -0.1% formic acid methanol solution as the mobile phase. Under optimized instrument conditions, electrospray positive ion multiple reaction monitoring (MRM) mode was employed for quantification using the external standard method, followed by methodological validation of the established approach. Results: The linearity of MAD in serum was good in the concentration range of 0.5-100 μg/L, and the correlation coefficient was 0.9997. The mean recoveries of MAD from spiked samples were 86.0%-109.6%, with the relative standard deviations were less than 10%. The limit of detection was 0.23 μg/L, The limit of detection was 0.75 μg/L. Conclusion: This method is high sensitive and reliable, which is suitable for the determination of MAD in in mouse serum.
    Keywords:  Chromatography, liquid; High performance liquid chromatography-tandem mass spectrometry; Maduramicin ammonium; Serum; Tandem mass spectrometry
    DOI:  https://doi.org/10.3760/cma.j.cn121094-20240518-00224
  21. Se Pu. 2025 Sep;43(9): 1034-1044
    [Rapid screening of 111 pesticides and veterinary drugs in livestock and poultry meat by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry].
    Yi-Ming Wang, Xiao-Tong Li, Kun Chu, Qian-Qian Wang, Shuai Wu, Chen Chen.
      The consumption of agricultural products has increased in recent years owing to abundant production and improved living standards. Veterinary drugs are highly commercialized and widely used in animal husbandry to ensure animal health and production performance. Moreover， pesticides can become enriched during animal breeding， resulting in animal-derived food pollution through foraging， drinking， and environmental disinfection that can potentially damage human health. Consequently， food-safety issues associated with pesticide and veterinary-drug residues have attracted considerable attention. However， few reports on the multi-residue analysis of livestock and poultry meat using ultra performance liquid chromatography-quadrupole time of flight mass spectrometry （UPLC-Q-TOF/MS） have been published. Therefore， developing high-throughput and efficient screening methods for monitoring various illegal and/or restricted drugs in animal-derived foods is imperative. In this study， we developed a protocol for simultaneously examining seven pesticide and veterinary-drug types that uses an accurate mass-spectral library. UPLC-Q-TOF/MS was then employed to screen 111 such compounds， including quinolones， macrolides， cephalosporins， and antiviral drugs. The developed protocol was subsequently used to establish a method for quantitatively analyzing more than 90 compounds in livestock and poultry meat. The formula， theoretical exact mass， experimental exact mass， and retention time of each analyte were recorded and used for identification purposes. The main factors affecting the response and sensitivity of the method， such as the LC separation conditions （chromatographic column and mobile phases） and MS parameters， were optimized during instrumental analysis. Pork and chicken samples were extracted with an 80% acetonitrile aqueous solution， after which the supernatant was purified using an Oasis PRiME HLB solid-phase extraction column. The 111 target analytes were separated on a Waters HSS T3 analytical chromatographic column （100 mm×2.1 mm， 1.8 μm） after being blown with nitrogen and redissolved， with gradient elution performed using mobile phases composed of 0.1% formic acid aqueous solution and methanol. The analysis process included a flow rate of 0.4 mL/min， a column temperature of 40 ℃， and an injection volume of 5 μL， with positive electrospray ionization （ESI） and time of flight mass spectrometry full scan information-dependent acquisition-product ion （TOF MS-IDA-Product Ion） scanning modes used. The method was validated in terms of linearity， limits of screening and quantification （SDLs and LOQs， respectively）， matrix effects， accuracy， and precision. Quantification was performed using matrix-matched external-standard calibration. All target compounds exhibited good linearities in their corresponding concentration ranges， with all correlation coefficients （r2） above 0.99. The SDLs of all analytes were in the range of 0.5-10 μg/kg， and the proportion of LOQs within the range of 0.5-10 μg/kg were 88.3% and 86.5%， respectively. The compounds quantified in pork and chicken exhibited recoveries of between 60.2% and 100.2%， and 61.1% and 116.7%， respectively， at spiked levels of LOQ， 2×LOQ and 10×LOQ， with relative standard deviations （RSDs） ranging from 1.1% to 13.9% and 1.0% to 14.1%， respectively. Simulated positive samples and commercial livestock and poultry meat samples were screened using an in-house-constructed mass spectrometry database. Commercial samples were screened while enrofloxacin was detected in two pork samples and tilmicosin was detected in one chicken sample， with content in the range of 4.94-29.1 μg/kg. The method developed in this study is advantageous because it involves simple sample processing and is less time consuming than existing methods； consequently， it is suitable for the rapid and high-throughput screening of pesticides and veterinary residues in livestock and poultry meat.
    Keywords:  livestock and poultry meat; multi-residue analysis; pesticides and veterinary drugs; rapid screening; ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry （UPLC-Q-TOF/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.10026
  22. Methods Mol Biol. 2025 ;2972 81-93
    Using HILIC-MS/MS Method to Study Inositol Pyrophosphate.
    Masatoshi Ito, Natsuko Fujii, Shinichi Koguchi, Eiichiro Nagata.
      The direct detection of InsP7 (diphosphoinositol pentakisphosphate) and InsP8 (bis-diphosphoinositol tetrakisphosphate), known as inositol pyrophosphates (PP-InsPs), in mammalian specimens faces technical difficulties owing to their characteristic chemical properties and minute quantities in mammalian tissues. We developed an analytical protocol to sensitively and directly detect PP-InsPs and their precursor, inositol hexakisphosphate (InsP6), using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). This analytical protocol, coupled with high-purity synthetic standards, allows for the absolute quantitation of these analytes in cellular samples as well as in various organs and tissues obtained by autopsies of animals and humans, offering an effective option to study PP-InsP functions in mammalian physiology and pathology. Herein, we describe this protocol in detail, from the sample preparation procedure throughout the column regeneration after analysis, along with several notable cautionary points.
    Keywords:  Hydrophilic interaction liquid chromatography; Inositol polyphosphate; Inositol pyrophosphate; InsP6; InsP7; InsP8; Liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-4799-8_7
  23. J Chromatogr A. 2025 Aug 24. pii: S0021-9673(25)00661-2. [Epub ahead of print]1760 466317
    Multi-mycotoxin determination in human urine by UHPLC-MS/MS: An environmentally friendly and high-throughput approach.
    Borja Peris-Camarasa, Olga Pardo, Pablo Dualde, Clara Coscollà.
      Mycotoxins, naturally occurring secondary metabolites produced by fungi, are globally prevalent in food and pose serious health risks to humans due to their toxicity, which include carcinogenic, mutagenic, and neurotoxic effects. Due to their diverse biochemical nature, these compounds follow distinct metabolic pathways in the human organism, with urinary excretion being their principal elimination route. Harmonization in human biomonitoring (HBM) is essential for comparable data across studies and regions, while trustworthy and validated analytical methodologies are vital for meaningful health insights. There's also a growing need for innovative, eco-friendly analytical techniques. In this context, we have developed and validated an eco-friendly analytical procedure for the rapid and simultaneous detection of multiple mycotoxins in urine. This approach utilizes a straightforward "dilute-and-shoot" sample preparation, combined with ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method targets twelve mycotoxins, achieving relative recoveries between 79.2 % and 113.5 %, with precision values consistently below 20 % for all analytes. The quantification range includes concentrations typically found in urine, with limits of quantification (LoQs) ranging from 0.005 to 0.5 ng/mL. Our evaluation demonstrates that this methodology offers a more sustainable and practical alternative to mycotoxin quantification in urine, with AGREEprep and BAGI scores of 0.73 and 85.0, respectively, and can be widely implemented in HBM programs. Furthermore, its application to 25 urine samples from adults residing in the Valencian Community (Spain) confirmed its effectiveness for HBM studies. Finally, a non-negligible level of exposure to these compounds within the population studied was observed.
    Keywords:  Dilute-and-shoot; Green analytical chemistry; Human urine; Mycotoxins; UHPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.chroma.2025.466317
  24. Analyst. 2025 Sep 03.
    Unsupervised machine learning for mass spectrometry imaging data analysis with in vivo isotope labeling.
    Raven L Buckman Johnson, Vy T Tat, Young Jin Lee.
      Mass spectrometry imaging (MSI) has emerged as a powerful tool for spatial metabolomics, but untargeted data analysis has proven to be challenging. When combined with in vivo isotope labeling (MSIi), MSI provides insights into metabolic dynamics with high spatial resolution; however, the data analysis becomes even more complex. Although various tools exist for advanced MSI analyses, machine learning (ML) applications to MSIi have not been explored. In this study, we leverage Cardinal to process MSIi datasets of duckweeds labeled with either 13CO2 or D2O. We apply spatial shrunken centroid (SSC) segmentation, an unsupervised ML algorithm, to differentiate metabolite localizations and investigate isotope labeling of untargeted metabolites. In the SSC segmentation of three-day 13C-labeled duckweed dataset, five spatial segments were identified based on distinct lipid isotopologue distributions, in contrast to classification of only three tissue regions in previous manual analysis based on galactolipid isotopologues. Similarly, SSC segmentation of five-day D-labeled dataset revealed five spatial segments based on distinct metabolite and isotopologue profiles. Further, this untargeted segmentation analysis of MSIi dataset provided insights on tissue-specific relative flux of each metabolite by calculating the fraction of de novo biosynthesis in each segment. Overall, the application of unsupervised machine learning to MSIi datasets has proven to significantly reduce analysis time, increase throughput, and improve the clarity of spatial isotopologue distributions.
    DOI:  https://doi.org/10.1039/d5an00649j
  25. Anal Bioanal Chem. 2025 Aug 30.
    Sustainable and renewable approaches in biomaterial-based integrated dried urine sampling/solid-phase extraction devices for drugs and metabolites: challenges, bioanalytical applications, and future directions.
    Sohan G Jawarkar, Pinaki Sengupta.
      Urine samples are gaining increasing importance as preferred biological matrices in bioanalytical studies due to their non-invasive collection, abundance, and ability to reflect systemic metabolism. However, conventional urine sampling and storage methods face significant challenges, especially in resource-limited areas where transportation and preservation infrastructure are inadequate. Traditional methods of liquid urine collection require refrigeration to prevent analyte degradation, making long-term storage and global sample transport costly and impractical. Biomaterial-based integrated dried urine sampling (DUS) devices integrate both storage and extraction into a single-step process, significantly enhancing efficiency and reliability. The porous sorbent is designed with a high surface area and tailored molecular binding capacity, ensuring the selective adsorption of target metabolites while minimizing matrix effects. Its structure allows for rapid drying and ambient-temperature storage, eliminating the need for cold-chain logistics. This feature is particularly beneficial for field studies and resource-constrained settings where maintaining refrigeration is impractical. Biomaterial-based DUS devices could be cost-effective and customizable and enable long-term stabilization of urinary metabolites. By reducing drying times, enhancing analyte recovery, and improving storage conditions, this technology enhances the feasibility of large-scale bioanalytical studies, therapeutic drug monitoring, and clinical diagnostics. Furthermore, its ability to selectively extract metabolites streamlines laboratory workflows and improves overall analytical precision. Porous biomaterial-based DUS devices could present a transformative, sustainable, and renewable solution for bioanalysis. By addressing the critical issues of sample stability, extraction efficiency, and cost-effectiveness, this approach has the potential to revolutionize metabolomics research, personalized medicine, and biomarker discovery in both clinical and research settings.
    Keywords:  Bioanalysis; Biomaterial-based matrix; DUS device; LC–MS/MS; Long-term storage and extraction; Urine metabolomics
    DOI:  https://doi.org/10.1007/s00216-025-06043-3
  26. Anal Chem. 2025 Sep 02.
    CLAW-MRM: Comprehensive Lipidomics Automation Workflow for Multiple Reaction Monitoring Using Large Language Models.
    Connor Beveridge, Sanjay Iyer, Caitlin E Randolph, Matthew Muhoberac, Palak Manchanda, Katherine A Walker, Shane Tichy, Gaurav Chopra.
      Lipidomic profiling generates vast datasets, making manual annotation and trend interpretation complex and time-intensive. The structural and chemical diversity of the lipidome further complicates the analysis. While existing tools support targeted lipid identification, they often lack automated workflows and seamless integration with statistical and bioinformatics tools. Here, we introduce the comprehensive lipidomics automated workflow for multiple reaction monitoring (CLAW-MRM), a platform designed to automate lipid annotation, statistical analysis, and data parsing using custom multiple reaction monitoring (MRM) precursor product ion transitions. CLAW-MRM employs trimmed mean of m-value (TMM) normalization to account for lipid load differences, enabling robust cross-sample comparisons. To evaluate CLAW-MRM's performance, we analyzed lipid profiles in liver tissues of Alzheimer's disease (AD) mice and age-matched wild-type controls under conditions of constant and variable tissue mass, assessing the impact of normalization strategies on TMM-normalized lipidomic outcomes. Additionally, we isolated and profiled lipid droplets from individual brain regions of 18- to 24-month-old AD male mice and controls, leveraging nearly 1,500 MRM transitions across 11 lipid classes. Enhancing biological relevance, CLAW-MRM integrates LIGER (lipidome gene enrichment reactions), linking lipid expression with gene activation and suppression patterns. Through CLAW-MRM-based LIGER, we identified metabolic pathways enriched in differentially expressed lipids, offering insights into altered lipid metabolism in AD. To improve usability, CLAW-MRM incorporates a natural language interface powered by large language models, enabling artificial intelligence (AI)-driven user interaction for statistical and bioinformatics analyses. By automating lipid structural identification and integrating AI-assisted bioinformatics, CLAW-MRM provides an end-to-end workflow from data acquisition to interpretation, streamlining high-throughput lipidomics.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05039
  27. Se Pu. 2025 Sep;43(9): 1045-1052
    [Simultaneous identification and detection of four novel isoxazoline drugs in bovine-origin foods using ultra-high performance liquid chromatography coupled to quadrupole/linear ion trap mass spectrometry].
    Cheng Yang, Wei-Xia Zhu, Ya-Feng Liu, Wei Wei, Fang Zhao, Kai Hu, Wen-Jie Zhao.
      Isoxazoline drugs （ISOs） are a class of five-membered heterocyclic compounds containing N and O atoms. They can inhibit γ-aminobutyric acid gated chloride channels and are widely used in the treatment of parasitic diseases in poultry. The intake of animal-derived foods by humans is an important way to come into contact with ISOs. Excessive use of ISOs can lead to their residues in animal-derived foods， thereby threatening human health and causing neurotoxicity and hepatotoxicity. To address the safety issues caused by ISO residues in animal-derived foods， an ultra-high performance liquid chromatography-quadrupole/linear ion trap mass spectrometry （UHPLC-Q/Trap MS） analytical method for four novel ISOs （fluralaner， sarolaner， afoxolaner， lotilaner） in bovine-origin foods （including milk， beef and bovine liver） was established. The sample was first extracted with acetonitrile and then purified with PRiME HLB solid phase extraction （SPE） column. Using 5 mmol/L ammonium acetate aqueous solution and acetonitrile as the mobile phase， after separation by Shim-pack GIST C18-AQ （100 mm×2.1 mm， 2.7 μm） chromatography column， the analysis was carried out in the multi-reaction monitoring （MRM） mode by information-dependent acquisition （IDA）， enhanced product ion scanning （EPI） and spectral library retrieval， and quantification was performed using the external standard method. The results showed that the four ISOs had good linear relationships within their respective mass concentration ranges. The correlation coefficients （r） were all ≥0.993 6， and the limits of detection （LODs） and quantification （LOQs） were 0.2-0.5 μg/kg and 0.5-1.0 μg/kg， respectively. Under the low， medium and high spiked levels （1， 2 and 10 μg/kg）， the recoveries of the four ISOs ranged from 67.6% to 118.9%， and the relative standard deviations （RSDs） ranged from 2.0% to 20.0%. In addition， in this study， qualitative screening and analysis of the target compounds were conducted through MRM-IDA-EPI combined with spectral library retrieval. Dual qualitative analysis of the target compounds was carried out based on information such as retention time and EPI fragment ions， which improved the accuracy of qualitative analysis and effectively eliminated the interference of false positive results. This method features low LODs and good recoveries. It is also simple and rapid to operate， with high sensitivity and accuracy. It can achieve qualitative and quantitative analysis of new ISOs residues in bovine-origin foods. This study can provide technical support for food safety agencies to implement preventive measures against new ISOs in animal foods.
    Keywords:  bovine-origin foods; fluralaner; isoxazoline drugs （ISOs）; solid phase extraction （SPE）; ultra-high performance liquid chromatography-quadrupole/linear ion trap mass spectrometry （UHPLC-Q/Trap MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.11012
  28. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 28. pii: S1570-0232(25)00324-1. [Epub ahead of print]1267 124770
    Development of a validated and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to simultaneous determination of crizotinib, alectinib and lorlatinib in human plasma.
    Haohua Chi, Shu Li, Fang Liu, Bo Li, Wenqian Chen, Cheng Zhang, Wei Zhou, Meng Yang, Pengmei Li.
      Crizotinib, alectinib, and lorlatinib are tyrosine kinase inhibitors used sequentially in non-small cell lung cancer (NSCLC) therapy. Therapeutic drug monitoring (TDM) is valuable during sequential treatment, enabling simultaneous quantification. Consequently, an analytical method capable of multiplexing these three compounds into a single assay is highly applicable for routine TDM. A multiplexed UHPLC-MS/MS method quantifying all three drugs was validated for selectivity, matrix effects, linearity (20-1000 ng/mL), accuracy, precision, carryover, and stability. Matrix effects were negligible in both plasma from healthy volunteers and plasma from patients with hyperlipidemia. No significant interfering responses were observed for crizotinib, alectinib, lorlatinib, or their corresponding isotope-labeled internal standards. Sample stability was confirmed under ambient conditions (25 °C) for at least 24 h. Furthermore, the pretreated supernatant remained stable in the auto sampler (15 °C) for 24 h. This validated method is suitable for application in pharmacokinetic studies and exposure-response assessments.
    Keywords:  Anaplastic lymphoma kinase inhibitors; Lorlatinib; Therapeutic drug monitoring; UHPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124770
  29. J Chromatogr Sci. 2025 Aug 30. pii: bmaf035. [Epub ahead of print]63(8):
    A Simple and Highly Sensitive Liquid Chromatographic-Mass Spectrometric Method for the Determination of Itraconazole and Its Major Metabolite in Human Plasma and Its Application to a Bioequivalence Study.
    Hamed Hamishehkar, Rasoul Hosseinpour, Ladan Dayani, Bahareh Samii, Jaber Emami.
      Itraconazole is an oral triazole antimycotic drug. Bioequivalence studies are cornerstones for the approval of generic drug development globally. The present study describes a simple, sensitive and economical LC-MS/MS method for the determination of itraconazole and its metabolite in human plasma. Itraconazole (drug), hydroxyitraconazole (metabolite), and atorvastatin as internal standard were added to plasma samples. The standard curve of both the drug and metabolite covering the 4-320 ng/mL concentration range, was linear (R2 = 0.999). Also, a limit of quantification of 4 ng/mL of a sample size of 0.4 mL is achieved which is comparable or even better than the reported methods. The applicability of this method was proven by analyzing true samples obtained after the administration of 100 mg itraconazole of test (Noxifunge®) and reference (Sporanox®) to healthy volunteers. The 90% confidence intervals of the logarithmically transformed AUC0-72, AUC0-∞, and Cmax of the two formulations both for the drug and the metabolite are within the accepted levels proposed by FDA and EMA. Therefore, the presented method is suitable for bioavailability, pharmacokinetic, and bioequivalent studies in humans.
    DOI:  https://doi.org/10.1093/chromsci/bmaf035
  30. J Am Soc Mass Spectrom. 2025 Sep 05.
    The Power of Many: An Ensemble Approach to Spectral Similarity.
    Javier E Flores, David J Degnan, Yuri E Corilo, Chaevien S Clendinen, Lisa M Bramer.
      Quantifying the similarity between two mass spectra─a known reference mass spectrum and an unidentified sample mass spectrum─is at the heart of compound identification workflows in gas chromatography-mass spectrometry (GC-MS). The reference spectrum most like the sample is assigned as its identification (provided some quantitative similarity threshold is met, e.g., 80%) and thus accurately measuring similarity is essential. Significant research has gone toward developing metrics for this purpose, each of which has attempted to improve upon existing methods by incorporating GC-MS-specific information (e.g., peak ratios or retention times) or adopting various statistical and algorithmic frameworks. While this active development has led to a plethora of similarity metrics with demonstrated value across different contexts, the unfortunate consequence has been confusion surrounding which metric should be used as a global standard. No such metric is currently accepted as the standard method because different metrics have demonstrated optimal performance in different contexts. In this work, we propose an ensemble approach to spectral similarity scoring that combines the collective information from across existing similarity metrics to form an improved, globally representative similarity metric as a step toward establishing a global standard method. The resulting ensemble metrics are evaluated on over 88,000 spectra of varying complexity and demonstrate improved abilities to accurately rank the correct reference spectrum as the top-matching candidate for a sample relative to the rankings generated by individual similarity scores.
    Keywords:  GC-MS; machine learning; mathematical methods; metabolite mixtures; metabolomics; spectral similarity
    DOI:  https://doi.org/10.1021/jasms.5c00176
  31. Anal Chim Acta. 2025 Oct 22. pii: S0003-2670(25)00815-3. [Epub ahead of print]1372 344421
    Investigation of morantel metabolism and its application in veterinary drug residue screening.
    Sedigheh Barzegar, Bryn O Shurmer, Anas El-Aneed, Randy W Purves.
       BACKGROUND: Food Safety authorities routinely test food of animal origin to verify that veterinary drug residues (VDRs) are within maximum residue limits. For morantel, a gas chromatography - mass spectrometry (GC-MS) confirmatory method is effective, but it involves an extensive sample preparation that is not suitable for multiple analytes. Furthermore, the resulting marker residue is not specific to morantel. Alternatively, using major morantel metabolites as specific VDRs in liquid chromatography - mass spectrometry (LC-MS) screening methods would increase productivity, especially when implemented in multi-residue screening methods.
    RESULTS: In this study, our workflow used in vitro incubations of morantel and stable isotope labeled morantel with liver S9 fractions. LC combined with high resolution mass spectrometry (HRMS) and Compound Discoverer software was used to identify major morantel metabolites. Detailed tandem MS (MS/MS and MS3) experiments were used to determine the structures of metabolites. Multiple phase I and phase II metabolites of morantel were identified, including hydroxylated and cysteine-conjugated metabolites. Five major metabolites, identified from in vitro metabolism studies using porcine or bovine liver S9 fractions, were thoroughly investigated. The presence of these metabolites was confirmed using pseudo-incurred bovine liver tissue. Metabolic reaction sites were proposed for these metabolites, including a hydroxylation site for one metabolite, that contradicts previous findings.
    SIGNIFICANCE: These five major metabolites reported are specific to morantel and can be readily implemented in a multi-residue screening method. This workflow is adaptable for other veterinary drugs and the major advantages of using this approach include time efficiency in sample preparation and the ability to incorporate the VDRs into multiple-residue screening methods.
    Keywords:  Food safety; HRMS; LC-MS/MS; Metabolite; Multi-residue; Semi-untargeted; Stable label isotope
    DOI:  https://doi.org/10.1016/j.aca.2025.344421
  32. Bioanalysis. 2025 Sep 04. 1-13
    Overcoming bioanalytical challenges during the development of risdiplam for the treatment of spinal muscular atrophy.
    Katja Heinig, Pawel Dzygiel, Luca Ferrari.
       AIMS: Risdiplam is a small molecule approved for the treatment of spinal muscular atrophy (SMA). The drug and its major metabolite had to be measured in plasma and tissue from several animal species and in human plasma and urine. Bioanalytical challenges including light sensitivity, instability, carryover, nonspecific binding, and complex tissue analysis, had to be overcome.
    MATERIALS & METHODS: Liquid chromatography tandem mass spectrometry with reversed-phase separation after protein precipitation/dilution was applied. Ascorbic acid was used as a stabilizer to mitigate degradation of the metabolite, and a surfactant additive prevented nonspecific binding in urine. Tissues were efficiently homogenized by bead beating and matrix-matched with plasma.
    RESULTS AND CONCLUSIONS: The above challenges were successfully addressed with bioanalytical methods tailored to study needs. Validations and regulatory analyses met requirements of current guidelines, including successful incurred sample reanalysis (ISR) in GLP and clinical studies. The 3R principles (Replacement, Reduction, Refinement) were applied in animal studies to minimize the use of real matrices. Pediatric studies were supported with rapid analysis and microsampling. Bioanalysis supported patient-centric approaches in dose finding and sampling and was key in answering important questions to enable risdiplam to the market.
    Keywords:  LC-MS/MS; Risdiplam; nonspecific binding; pediatrics; stabilization; tissue
    DOI:  https://doi.org/10.1080/17576180.2025.2554563
  33. Anal Sci Adv. 2025 Dec;6(2): e70038
    Rapid Dereplication of Bioactive Compounds in Plant and Food Extracts Using Liquid Chromatography-Electrospray-Tandem Mass Spectrometry.
    Naheed Akhtar, Adeeba Khadim, Syed Usama Yaseen Jeelani, Bibi Zareena, Arslan Ali, Jalal Uddin, Hesham R El-Seedi, Satyajit D Sarker, Muhammad Ramzan, Syed Ghulam Musharraf.
      High-throughput screening and identification of common phytochemicals are crucial for lead optimization, drug development and investigation of metabolic pathways in complex herbal extracts. The available databases contain a huge number of compounds, making it challenging and time-consuming to dereplicate valuable compounds. Therefore, the current study aimed to develop an in-house mass spectral library for the rapid dereplication of 31 commonly occurring natural products from different classes using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). A total of 31 standards were grouped into two different pools, and each pool was analysed under uniformly optimized conditions in positive ionization mode. A pooling strategy on the basis of log P values and exact masses was adopted to minimize the co-elution and the presence of isomers in the same pool. The MS/MS features of each compound were acquired using [M + H]+ and/or [M + Na]+ adducts at 25.5-62 eV range as average collision energy and 10, 20, 30 and 40 eV as individual collision energies. The names, molecular formulae, exact masses with <5 ppm error, MS and MS/MS features of analysed reference compounds were used to construct the MS/MS library. The developed MS/MS library was efficiently used for the rapid dereplication and validation of 31 compounds in 15 different food and plant sample extracts. The MS data of 31 reference standards have been submitted to the MetaboLights online database (MTBLS9587). The developed library will be beneficial for the rapid dereplication of biologically valuable compounds in a variety of herbal formulations and food samples.
    Keywords:  LC–HR–ESI–MS/MS; dereplication; flavonoids; tandem mass spectral library; triterpenes
    DOI:  https://doi.org/10.1002/ansa.70038
  34. Anal Chem. 2025 Aug 31.
    Large Language Model-Empowered Compound Collision Cross-Section Prediction.
    Zeyu Zhu, Chengyi Xie, Shaojie Lin, Xiangnan Xu, Shuxian Niu, Lei Guo, Jiyang Dong.
      Collision cross section (CCS) is a crucial parameter in ion mobility-mass spectrometry, which plays a significant role in enhancing the precision of compound annotation. Computational prediction methods aim to infer the CCS value from molecular structure and have become a common strategy for efficiently building large-scale CCS compound databases. However, most of the current available methods deliver suboptimal predictive performance due to limited high-quality training data sets and inadequate model architectures for handling multimodal features. To address these issues, we present HyperCCS, a novel CCS prediction framework powered by chemical large language models (CLLMs). Through fine-tuning a CLLM that has been trained on billions of SMILES sequences, HyperCCS effectively captures complex structural and chemical information that the current models trained on a limited CCS data set might miss. A cross-modal feature fusion module is designed to dynamically integrate CLLM-derived features with other heterogeneous features, effectively resolving structural ambiguities commonly found in multimodal features. Benchmark evaluation on the METLIN-CCS and AllCCS2 data sets shows that HyperCCS achieves robust CCS prediction on molecules of various masses, adduct types, and ion modes, outperforming other methods. To showcase the adaptability of HyperCCS within a real-world system, results on in-house experimental data demonstrate its ability to accurately resolve isomers and extrapolate to high-mass analytes. In addition, SHAP analysis and ablation studies confirm the crucial role of CLLM-derived features and the cross-modal feature fusion mechanism in enhancing CCS prediction. HyperCCS is anticipated to offer a high-throughput and cross-instrument computational tool to aid experimental efforts in metabolomics and structural biology.
    DOI:  https://doi.org/10.1021/acs.analchem.5c03492
  35. NMR Biomed. 2025 Oct;38(10): e70131
    Automatic Identification of Potential Cellular Metabolites for Untargeted NMR Metabolomics.
    Jiashang Chen, Angela Rao, Rajshree Ghosh Biswas, Ella J Zhang, Jonathan Xin Zhou, Evan Zhang, Zuzanna Kobus, Marta Kobus, Li Su, David C Christiani, David S Wishart, Leo L Cheng.
      An organism's metabolic profile provides vital information pertaining to its physiology or pathology. To monitor these biochemical changes, Nuclear Magnetic Resonance (NMR) spectroscopy has found success in non-invasively observing metabolite changes within intact samples in an untargeted manner. However, biological samples are chemically complex, comprised of many different constituents (amino acids, carbohydrates, and lipids) at varying concentrations depending on physiological and pathological conditions. Due to the narrow spectral window of proton NMR, compound resonance frequencies can often overlap, making the identification and monitoring of metabolites difficult and time consuming, particularly when dealing with large numbers of samples. Here, we introduce a Python program (ROIAL-NMR) to systematically identify potential metabolites from defined proton NMR spectral regions-of-interest (ROIs), which are identified from complex biological samples (i.e., human serum, saliva, sweat, urine, CSF, and tissues) using the Human Metabolome Database (HMDB) as a reference platform. Briefly, for disease-versus-control studies, the program considers disease types and utilizes study-defined ROIs together with their differing intensity levels, according to sample types, in differentiating disease from control to propose potential metabolites represented by these ROIs in an output table. In this report, we illustrate the utility of the program with one of our recent studies, where we measured proton NMR spectra of serum samples taken from lung cancer (LC) patients, with and without Alzheimer's disease and related dementia (ADRD). The program successfully identified 88 metabolites, with 66 differentiating LC from control patients, and 80 distinguishing LC patients with ADRD from those without ADRD to provide important information regarding pathophysiology in complex biological samples.
    Keywords:  automated metabolite identification; nuclear magnetic resonance spectroscopy; python program
    DOI:  https://doi.org/10.1002/nbm.70131
  36. J Chromatogr A. 2025 Aug 07. pii: S0021-9673(25)00621-1. [Epub ahead of print]1760 466276
    Miniaturized one-step extraction for the analysis of tetrahydrocannabinol ∆9- and ∆8- isomers, phytocannabinoids, and hexahydrocannabinol in whole blood using liquid chromatography tandem mass spectrometry.
    Manuela Carla Monti, Anna Stoll, Götz Schlotterbeck, Urs Duthaler.
      ∆9-tetrahydrocannabinol (∆9-THC) plays a major role in diving under the influence of drugs investigations and workplace drug testing. Additionally, cannabidiol (CBD), as well as semi-synthetic cannabinoids, like hexahydrocannabinol (HHC) and ∆8-tetrahydrocannabinol (∆8-THC), are increasingly found on regulated and unregulated drug markets. A straightforward bioanalytical method was developed, covering 14 analytes, including ∆9-THC, CBD, ∆8-THC, and their respective metabolites, as well as HHC and a subset of minor phytocannabinoids. Sample cleanup consisted of one precipitation step conducted in a 96-well plate format and requiring only 50 µL of whole blood. For analysis an ultra high-performance liquid chromatography system coupled to a triple quadrupole mass spectrometer was used. The method was validated in accordance with ICH M10 and guidelines in forensic toxicology. The method proved to be precise and accurate with a maximal inter assay imprecision of 9.4 % and a maximal accuracy bias of 12.3 %. The validation revealed good recoveries (>86 %), highly consistent matrix effects (relative standard deviation; RSD ≤7 %) and suitable limits of quantification (LOQs), i.e. 0.5 µg/L for ∆8- and ∆9-THC. In addition, baseline separation of ∆8-THC-COOH and ∆8-THC from the corresponding ∆9-THC isomers were achieved. Method validation demonstrated a fit for purpose analytical method covering legal thresholds for ∆9-THC for driving. Samples from 4 individuals after inhalation of CBD-rich cannabis were analyzed, whereby 8 out the 14 analytes could be quantified, demonstrating the utility and suitability of the method. The method reduces sample volume, solvent use, and workload, making it well-suited for automation and clinical or forensic applications.
    Keywords:  Cannabis; DUID; Forensic toxicology; HHC; Semi-synthetic cannabinoids
    DOI:  https://doi.org/10.1016/j.chroma.2025.466276
  37. Se Pu. 2025 Sep;43(9): 1063-1069
    [Rapid determination of five antipyretic analgesics in surface water by online solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry].
    Ping He, Li-Qun Wang, Shan Zhou, Bao-Feng Zhang, Xuan Jia, Yi Chi, Zhen-Qi Xu, Wei Tang.
      Antipyretic analgesics are typical pharmaceutical and personal care products （PPCPs） that are widely used in our daily life because they relieve fever and pain， and have anti-inflammatory and anti-rheumatic properties. These drugs inhibit the synthesis and release of prostaglandins （PGs） in the neurons of the anterior hypothalamus and exert therapeutic effects as a consequence. However， these drugs are relatively commonly misused and abused， often owing to a lack of proper medication guidance. As a result， these drugs enter the environment via various pathways， including wastewater treatment plants， agricultural runoff， and improper disposal， thereby posing potential threats to human health and ecosystems. The presence of these contaminants in surface water has become an environmental safety concern that necessitates the development of rapid， accurate， and high-throughput analysis methods. In this study， an analytical method was established for the determination of five antipyretic analgesics （ibuprofen， aminophenazone， antipyrine， phenacetin， and naproxen）. The developed method is based on online solid phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry （online SPE-UHPLC-MS/MS）， which provides a high degree of automation and efficiency. Water samples were collected and filtered through 0.2-μm regenerated cellulose （RC） filter membranes， after which Na2EDTA and an internal standard were added. An aliquot （0.9 mL） of each sample was injected into the online SPE system using an automatic sampler. Samples were first adsorbed on a PLRP-S online SPE column， washed with 0.05% formic acid aqueous solution， and finally gradient-eluted with a mobile phase composed of 0.2 mmol/L ammonium fluoride solution and methanol-acetonitrile （1∶1， v/v）. Analytes were separated on a ZORBAX Eclipse Plus C18 column， detected by multiple reaction monitoring with electrospray ionization in both positive- and negative-ion modes， and quantified using the internal-standard method.The five antipyretic analgesics were effectively separated under the optimized experimental conditions and showed good linearities within their respective concentration ranges， with correlation coefficients （r） greater than 0.998. The method detection limits （MDLs） ranged from 0.05 to 0.20 ng/L， and the method quantification limits （MQLs） ranged from 0.20 to 0.80 ng/L. The five antipyretic analgesics exhibited average recoveries of between 64.2% and 112%， with relative standard deviations （RSDs， n=6） of 2.06%-8.99% at low， medium， and high spiked levels. Furthermore， the method was successfully used to analyze water samples from the Hangzhou section of the Qiantang River， in which four target compounds were detected， with antipyrine found to have the highest mass concentration. This newly developed method features a high degree of automation， facilitates the injection of large volumes， and enables online enrichment， purification， and quantitative analysis in an integrated process， with minimal operational errors， high repeatability， and high sensitivity. The developed method shortens sample-analysis times， is more cost-effective， and meets the needs of large-scale sample testing， as it requires only 15 min to completely determine a sample， thereby significantly improving detection efficiency. Overall， the method features low detection limits， analyzes rapidly， and is easy to operate； hence it is suitable for rapid risk screening and the quantitative determination of antipyretic analgesics in surface water.
    Keywords:  antipyretic analgesics; online solid phase extraction （online SPE）; surface water; ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.10018
  38. Methods Mol Biol. 2025 ;2972 95-113
    Capillary Electrophoresis Mass Spectrometry for Inositol (Pyro)phosphate Profiling.
    Guizhen Liu, Henning J Jessen.
      The turnover of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a dynamic process that plays an important role in many physiological processes by transmitting signals within cellular pathways and networks. Profiling the InsPs and PP-InsPs isomers and quantifying their change in abundance is a significant challenge for several reasons. First, InsPs and PP-InsPs constitute a diverse metabolite pool, characterized by the complexity as a result of the numerous possible isobaric isomers. Second, these species are usually of low abundance in biological samples. Third, they lack a chromophore, making UV or fluorescence detection unfeasible. Fourth, their high charge density and the instability of P-anhydride bonds make isolation and separation requirements particularly demanding. This chapter presents a capillary electrophoresis coupled to mass spectrometry (CE-MS) method as a powerful tool. It enables the separation of multiply charged InsPs and PP-InsPs with high resolution for profiling regioisomers with high sensitivity from biological samples.
    Keywords:  Capillary electrophoresis; Inositol phosphate; Inositol pyrophosphate; Mass spectrometry; Phospho-metabolomics
    DOI:  https://doi.org/10.1007/978-1-0716-4799-8_8
  39. J Am Soc Mass Spectrom. 2025 Sep 01.
    Profiling Multiple Steroid Subclasses Enabled by Rounded Turn SLIM Ion Mobility.
    Cole L Frank, Breland M Jones, Emmaleigh Efird, Makenna Hoover, Selena Kingsley, Heidi M Sabatini, Christopher D Chouinard.
      Ion mobility-mass spectrometry has shown tremendous potential for improving the analysis of various subclasses of steroids. Its speed and ability to separate isobaric and isomeric species makes it ideal for biomedical, clinical, food, environmental, and antidoping analyses. But while other high-resolution ion mobility (HRIM) techniques have begun to see increased use in steroidomics, Structures for Lossless Ion Manipulations (SLIM) is a relative newcomer to the field. HRIM methods are generally challenged with low molecular weight (MW < 400) analysis, but a recently developed SLIM platform has incorporated rounded turns, helium buffer gas, and increased SLIM RF frequency to enable improved low mass transmission. In the present work, we investigate this low mass-tailored SLIM system for the analysis of anabolic androgens, bile acids, endocrine hormones, and ketosteroids. Linear regression-corrected SLIMCCSHe (CCS = collision cross section) values show excellent agreement with values obtained via direct measurement with a drift tube instrument but also underlie the importance of using class- and adduct-specific correction factors. Next, separation performance was evaluated for a range of challenging isomers and demonstrated good resolution for species with ΔCCS of <1%. Finally, the new rounded turn system was compared quantitatively with the traditional 90° (square turn) system in both nitrogen and helium buffer gases and revealed significantly improved signal across the board for rounded turn analyses with helium. Overall, this low mass-tailored system has remarkable promise for development of rapid, targeted steroid analysis in a range of applications.
    Keywords:  Ion Mobility-Mass Spectrometry; Steroids; Structures for Lossless Ion Manipulations (SLIM)
    DOI:  https://doi.org/10.1021/jasms.5c00218
  40. J Chromatogr Sci. 2025 Aug 30. pii: bmaf047. [Epub ahead of print]63(8):
    Simultaneous Analysis of Cyproconazole and Tebuconazole in Suspension Concentrate Formulation by Reverse-Phase High-Performance Liquid Chromatography.
    Sahadev Katam, Prasad Munipalli, Bhavani Balram, Vasu Govardhana Reddy Peddiahgari.
      A novel and validated reverse-phase high-performance liquid chromatography (RP-HPLC) approach was established for the concurrent measurement of cyproconazole (CYP) isomers and tebuconazole (TBZ) in suspension concentrate (SC) agrochemical formulations. The approach employed a C18 column with a gradient elution of 0.1% formic acid in water and methanol, attaining baseline resolution of CYP isomer-1, isomer-2 and TBZ without requiring chiral columns or sample pretreatment. Linearity was confirmed within the range of 80-120% of the target concentration, with a R2 value of 0.999. Accuracy varied from 101.2% to 102.8%, with a %RSD of less than 0.8%. LC-MS analysis verified analyte identity through distinctive ion transitions (CYP: m/z 292, TBZ: m/z 308). This is the initial method for isomer-specific detection of CYP using RP-HPLC within a formulation matrix. The methodology is straightforward, resilient and appropriate for standard quality assurance and regulatory adherence in agrochemical manufacturing.
    DOI:  https://doi.org/10.1093/chromsci/bmaf047
  41. Metabolomics. 2025 Aug 29. 21(5): 126
    Combining clinical chemistry with metabolomics for metabolic phenotyping at population levels.
    Yun Xu, Ian D Wilson, Royston Goodacre.
       INTRODUCTION: Untargeted metabolic phenotyping (metabolomics/metabonomics), also known as metabotyping, has been shown to be able to discriminate reliably between different physiological or clinical conditions. However, we believe that standard panels of routinely collected clinical and clinical chemistry data also have the potential to provide assay panels that complement metabotyping.
    OBJECTIVES: To test the above hypothesis and evaluate the use of multivariate statistical analyses to provided panels of clinical/clinical chemistry data measurements that predict the age, sex and body mass index (BMI) of 977 normal subjects and compare these predictions with results acquired by metabotyping on the same healthy individuals.
    METHODS: Metabotyping involved serum metabolomics using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) previously reported in our HUSERMET study (Dunn et al., 2015), while clinical chemistry data were obtained in clinic for 19 measurements assessing liver and kidney function, blood pressure, serum glucose, cations, as well as lipids. Multivariate analyses involved using support vector machines, random forest and partial least squares, to predict sex, age and BMI. These models used as inputs: (i) the clinical chemistry data alone; (ii) three metabolomics datasets; (iii) combinations of clinical chemistry with the metabolomics data. Model predictions were rigorously validated using 1,000 bootstrapping re-sampling coupled with permutation tests.
    RESULTS: Multivariate statistical analyses on the clinical chemistry data obtained for these healthy participants could be used to predict: their sex, based on creatinine; their age, based on systolic blood pressure, total serum protein and serum glucose; as well as BMI using alanine transaminase, total cholesterol (Total-c) to high-density lipoprotein cholesterol (HDL-c) ratio and diastolic blood pressure. Combining clinical chemistry and metabolomics data sets enhanced the predictions of these characteristics. Moreover, this powerful combination allowed for quantitative predictions of age and BMI.
    CONCLUSION: Multivariate statistical analysis on clinical chemistry data from the HUSERMET study obtained similar predictions of age, sex or BMI, compared to metabotyping using GC-MS and LC-MS. These predictions from clinical chemistry data were between 71 and 85% accurate (depending on the MVA used) and compared favourably with metabolomics (71-91 depending on analytical method). Combining clinical chemistry and metabolomics data sets enhanced the predictions of these characteristics to 77-93% accuracy, suggesting that this augmentation of methods may be a useful approach in the search for clinical biomarkers.
    Keywords:  Biomarker discovery; Clinical chemistry; HUSERMET study; Metabotyping; Multivariate statistical analysis
    DOI:  https://doi.org/10.1007/s11306-025-02331-2
  42. J Am Soc Mass Spectrom. 2025 Sep 04.
    High-Specificity and Sensitivity Imaging of Neutral Lipids Using Salt-Enhanced MALDI TIMS.
    Kameron R Molloy, Martin Dufresne, Madeline E Colley, Lukasz G Migas, Raf Van de Plas, Jeffrey M Spraggins.
      Neutral lipids are vital to various cellular processes and disease pathologies. However, their characterization by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) remains challenging due to poor ionization efficiency and difficulties distinguishing subtle structural differences among numerous isomeric and isobaric species. In this study, we enhanced neutral lipid detection by incorporating isotonic metal-cation washes into our MALDI IMS sample preparation workflow. Resulting salt adducts improved neutral lipid isobar and isomer separation by using trapped ion mobility spectrometry (TIMS). This approach increased both sensitivity and specificity for neutral lipid IMS experiments across multiple organ types, including murine brain, rabbit adrenal gland, human colon, and human kidney. Comparative analyses revealed that the most effective salt wash was tissue-dependent. However, the Na+ carbonate buffer solution (CBS) wash showed the greatest overall increase in neutral lipid detection. These findings provide a robust framework for mapping neutral lipids across multiple tissues and disease states and allow for the detailed characterization of neutral lipid isomers and isobars in complex biological tissues.
    DOI:  https://doi.org/10.1021/jasms.5c00202
  43. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Aug 21. pii: S1570-0232(25)00319-8. [Epub ahead of print]1266 124765
    Development of a pseudotargeted metabolomics approach for relative quantification of free fatty acid double-bond isomers in beagle plasma.
    Wen Xiao, Haihui Ren, Yueyi Zhang, Jiameng Han, Qiaorong Liu, Zunjian Zhang, Yuan Tian.
      Free fatty acids (FFAs) play a key role in living organisms and participate in metabolic processes, mainly as a source of energy for cells. The CC position isomer of FFAs is strongly associated with many diseases, but quantification of its double bond position isomer remains a challenge in lipid analysis. In this study, based on the requirements of the pseudotargeted metabolomics, an analytical method based on a two-step derivatization LC-MS/MS method and a multiple reaction monitoring (MRM) mode was established and fully validated in terms of linearity, precision, and stability. The results demonstrated that the method relatively quantified 30 FFAs in plasma samples. These included 8 saturated fatty acids (SFAs), 12 monounsaturated fatty acids (MUFAs), and 10 polyunsaturated fatty acids (PUFAs). The developed method was applied to analyze the increase or down-regulation of the relative content of FFAs before and after the administration of the drug in beagles which may help to explain the therapeutic effect of sacubitril valsartan. The establishment of this method allows the study of FFAs in beagles plasma to be no longer limited to analysis at the subclass level but can be focused at the isomer level.
    Keywords:  FFAs; Isomer; LC-MS/MS; Pseudotargeted Metabolomics; Relative Quantification; Two-step Derivatization
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124765
  44. Se Pu. 2025 Sep;43(9): 1053-1062
    [Determination of 79 pesticide residues in fruits and vegetables by QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry].
    Le Zhao, Xian-Jun Liu, Hao Zhang, Jian Li, Liang Cai, Xiang Fan, Tan-Yao Li, Dong-Yang Chen.
      Pesticide residues in fruits and vegetables are becoming a serious issue. These residues can affect the quality of agricultural products and people's health. Therefore， it has become crucial to effectively monitor and control pesticide residues in the food safety field. In this study， a rapid and effective QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry （QuEChERS-UPLC-MS/MS） method was established for the simultaneous determination of 79 typical pesticides in vegetables and fruits， including organophosphates， carbamates， and pyrethroids. The pretreatment， UPLC， and MS/MS conditions were optimized. The fruit and vegetable samples were extracted with frozen acetonitrile after pulverization and homogenization， cleaned up by the QuEChERS method， filtered through a centrifugal membrane， and analyzed by UPLC-MS/MS. The separation was carried out on an ACQUITY UPLC HSS T3 column （100 mm×2.1 mm， 1.8 μm） with gradient elution. The aqueous and organic phases were water-methanol （98∶2， v/v） and methanol-water （98∶2， v/v） respectively， both with 5 mmol/L ammonium acetate and 0.1% formic acid. A triple quadrupole mass spectrometer was used in positive-ion electrospray ionization （ESI+） scanning mode， with target pesticide residues quantified using the matrix-matched standard-curve method. The results showed that under the optimized conditions， the 79 target compounds were determined with good linearities in the range of 0.1-200 μg/L， and the correlation coefficients （r） were all greater than 0.990. The limits of detection （LODs） and limits of quantification （LOQs） of the 79 compounds were in the range of 0.01-4.0 μg/kg and 0.03-13.0 μg/kg. The recoveries at three spiked levels ranged from 78.2% to 119.8%， with relative standard deviations （RSDs） all less than 15.8%. The established method was successfully applied to 80 samples of fruits and vegetables from Hunan province. As a result， 19 pesticides were detected in 31 samples， and thiamethoxam， acetamiprid and clothianidin being the most highly detected with a content range of 0.012-2.62 mg/kg； According to the data of the Hunan province survey yearbook， the percentages of acceptable daily intake （%ADI） for chronic dietary exposure of the detected neonicotinoid insecticides （thiamethoxam and clothianidin） have been calculated. The results indicate that the %ADI of clothianidin in fruits and vegetables ranged from 5.74% to 0.36%， respectively， and the %ADI of thiamethoxam in fruits and vegetables ranged from 0.40% to 19.50%. The %ADI of both pesticides were found to be less than 100%， indicating they are within acceptable limits. The method is simple， sensitive， accurate， and suitable for the simultaneous determination of multiple pesticide residues in fruits and vegetables.
    Keywords:  QuEChERS; pesticide residues; risk assessment; ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）
    DOI:  https://doi.org/10.3724/SP.J.1123.2025.02015
  45. Bull Exp Biol Med. 2025 Aug 29.
    Analysis of Serum Serotonin by High-Performance Liquid Chromatography-Tandem Mass Spectrometry Using Online Solid-Phase Extraction.
    D V Bychenkov, V A Ioutsi, I V Kirilina, M V Ovcharov, M A Antsupova, A K Lubenskaya, L K Abdullaev, S A Pantsernaya, N G Mokrysheva.
      We propose a method for the quantitative determination of serotonin in blood serum using high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) and online solid-phase extraction in a chromatographic system. This approach allows serotonin quantification in blood serum within a range of 0.5-2000 ng/ml, partially automates the sample preparation process, and minimizes the influence of matrix components. The method is designed for the quantitative measurement of serotonin levels in patients with various deviations of serotonin levels and associated diseases.
    Keywords:  high-performance liquid chromatography; online solid-phase extraction; quantitative analysis; serotonin; tandem mass spectrometry
    DOI:  https://doi.org/10.1007/s10517-025-06474-z
  46. J Chromatogr A. 2025 Aug 13. pii: S0021-9673(25)00639-9. [Epub ahead of print]1760 466294
    Development and application of a quantitative Liquid chromatography-tandem mass spectrometry method for multi-class organic contaminants in biological and environmental matrices using a desirability function-based optimization.
    Lounes Haroune, Sabrina Saibi, Olivier Savary, Hubert Cabana, Jean-Philippe Bellenger.
      The diversity of trace organic contaminants (TrOCs) in complex matrices makes analytical development challenging. This study aimed to develop, optimize and validate an analytical method for extracting and quantifying 71 TrOCs. The development was performed using a factorial design with the desirability function as a quantitative tool for method optimization. TrOCs were extracted using a salt-out approach with ethyl acetate/water (4/1) and cleaned up by dispersive solid-phase extraction (C18/Na2SO4) before LC-MS/MS analysis. The method exhibited a high Average Overall Recovery (AOR up to 91%), with low limits of detection (from 0.12 to 1.79 ng g-1) and quantification (from 0.42 to 4.72 ng g-1). The evaluation of matrix effects revealed predominantly weak ionic suppression (-16% on average). The applicability and robustness of the validated method were demonstrated through the analysis of real complex environmental samples, including municipal biosolids, sediment samples and benthic organisms (Chironomidae and Oligochaete) from Canadian Lake. The results highlighted the effectiveness of the desirability function as a quantitative tool for selecting optimal conditions when multiple parameters need to be considered. This approach provided valuable insights supporting decision-making during method development, where regular statistical analysis did not reveal significant differences between conditions. The developed method provides a reliable tool for monitoring TrOCs across various environmental matrices, facilitating quantitative assessment and management of environmental risks.
    Keywords:  Biosolids; Desirability function; Environmental samples; Liquid chromatography; Mass spectrometry; Organic contaminants; Pesticides; Pharmaceutical compounds
    DOI:  https://doi.org/10.1016/j.chroma.2025.466294
  47. Anal Chem. 2025 Sep 02.
    Metabolite Fusion between Breath and Blood Enables More In-Depth Understanding of the Endogenous Metabolome.
    Zhifeng Tang, Jianming Yang, Bingtian Su, Xin Xu, Xin Luo, Huiling Wang, Keda Zhang, Tao Huan, Pablo Sinues, Mingliang Fang, Xue Li.
      Blood is a widely used sample type in metabolomics but often loses volatile compounds during analysis. In contrast, exhaled breath offers a noninvasive and complementary matrix that retains these volatiles. However, the accuracy of metabolite identification in breath remains a key challenge. To address this, we developed and integrated three novel strategies to enhance the characterization of the human metabolome: (1) a controlled exercise protocol was applied to capture biologically relevant metabolic changes, (2) water-gas partition coefficients and a self-built breath metabolomics database were incorporated to enhance the identification accuracy of breath metabolites, and (3) fusion of breath and blood metabolites was conducted to expand metabolite coverage and validate the reliability of breath metabolite identification. Using high-resolution tandem mass spectrometry, we conducted an untargeted metabolomics analysis of breath and blood samples collected during exercise. A total of 66 metabolites were uniquely identified in breath, 59 were unique to blood, and only 4 were shared between the two. Fusion of breath and blood data expanded the coverage of exercise-associated metabolic pathways and revealed breath-specific markers of exercise-induced metabolic changes. This study presents an accurate and integrative strategy for breath metabolite discovery, advancing our understanding of in vivo metabolism and offering promising biomarkers for smart wearable devices.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00543
  48. J Pharm Biomed Anal. 2025 Aug 27. pii: S0731-7085(25)00470-4. [Epub ahead of print]267 117129
    Unraveling the bridging region of Lenacapavir atropisomers by two-dimensional liquid chromatography.
    Yi Shen, Sarju Adhikari, Gavin Carr, Chris Foti, Alban R Pereira.
      Atropisomers are stereoisomers that arise from restricted bond rotation and undergo dynamic conformational change depending on time and temperature. The interconversion of atropisomers could result in a non-baseline "plateau" or "bridging area" between the isomer peaks in liquid chromatography (LC), which diminishes the chromatographic resolution and reduces the assurance of the analyte's purity. Conventional one-dimensional LC (1D-LC) separation relies on one or two major interactions among the analyte, mobile and stationary phase in an analytical column, which may not be sufficient for deciphering the bridging area. In contrast, two-dimensional LC (2D-LC) leverages orthogonal separation mechanisms in two dimensions, significantly enhancing peak capacity to resolve co-eluting impurities and ensure peak purity. Herein, we present a comprehensive 2D-LC study to investigate the on-column interconversion of Lenacapavir (LEN), which exists as a mixture of two atropisomers. Firstly, different columns, temperature, mobile phase pH, and cation additives were investigated using 1D-LC to assess their potential effects toward on-column interconversion of LEN atropisomers. The bridging area of LEN atropisomers was then unraveled using the heart-cutting mode in 2D-LC, presenting varying ratios for the two atropisomers across the retention time window of LEN. Furthermore, orthogonal conditions for the second dimension were developed to disentangle possible impurities in the bridging area using a regioisomer as surrogate. Lastly, and most importantly, by implementing the developed second-dimension methods, a co-eluting regioisomer of LEN can be separated using 2D-LC. With the aid of high-resolution mass spectrometry (HRMS), we were able to distinguish the co-eluting regioisomer down to 0.05 % (w/w).
    Keywords:  2D-LC; Atropisomer; On-column interconversion; Regioisomer LC separation; Tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.jpba.2025.117129
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