bims-metlip 2025-02-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2025–02–23
thirty-two papers selected by
Sofia Costa, Matterworks

A Sensitive and Selective LC-MS/MS-ESI Method for the Quantitation of Metabolites M9, M12, and M20 of Bexicaserin in Human Plasma and Urine Matrices.
Simultaneous Quantification of Filgotinib and Its Active Metabolite in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry: Validation and Clinical Application.
From Complexity to Clarity: Expanding Metabolome Coverage With Innovative Analytical Strategies.
A systematic analysis of in-source fragments in LC-MS metabolomics.
SimMS: A GPU-Accelerated Cosine Similarity implementation for Tandem Mass Spectrometry.
Comprehensive tissue homogenization and metabolite extraction for application in clinical metabolomics.
Part A: Implementing an Analyte Panel and Sampling Protocol for Quality Control in Mass Spectrometry Imaging.
A dilute-and-shoot LC-MS/MS determination of low-dosage third-generation antipsychotics and their metabolites in urine using an ultra-short column.
Quantitation of antibiotics in fresh fermentation medium by hydrophilic interaction chromatography mass spectrometry.
Quantification of Pectolinarin in Rat Plasma Using UPLC-MS/MS and Its Pharmacokinetic Analysis.
Salting-out assisted liquid-liquid extraction for UPLC-MS/MS determination of bile acids and kynurenine-, indole- and serotonin-pathway metabolites of tryptophan in human serum of healthy probands.
In-situ formation of deep eutectic supramolecule based extraction method coupled to valve switching ion chromatography mass spectrometry for the determination of aminoglycosides in meat.
From Reverse Phase Chromatography to HILIC: Graph Transformers Power Method-Independent Machine Learning of Retention Times.
Simultaneous Determination of Multiple Acid-Suppressing Drugs by UPLC-MS/MS Method and Application for Pharmacokinetics Study.
Comparison of Mid-Infrared and Ultraviolet Lasers Coupled to the MALDESI Source for the Detection of Secondary Metabolites and Structural Lipids in Arabidopsis thaliana.
Phosphorylated glycosphingolipids are commonly detected in Caenorhabditis elegans lipidomes.
Research on similarity retrieval method based on mass spectral entropy.
High throughput analysis of vancomycin in human plasma by UHPLC-MS/MS.
Successive electromembrane extraction: A new insight in simultaneous extraction of polar and non-polar metabolic molecules from biological samples.
Performance Comparison of Liquid Chromatography and Paper Spray Ionization with Mass Spectrometry for Measuring Kinase Inhibitors in Human Plasma.
An automated liquid-liquid extraction platform for high-throughput sample preparation of urinary phthalate metabolites in human biomonitoring.
Validation of a quantitation method for conjugated quercetin in human plasma.
Analytical performance evaluation and optimization of serum 25(OH)D LC-MS/MS measurement.
Recent advances in lipid analysis by capillary electromigration methods, 2019-2024.
Proteoform identification using multiplexed top-down mass spectra.
A sensitive LC-MS/MS method for the quantification of serum epoxyeicosatrienoic and dihydroxyeicosatrienoic acids in the identification of diabetic kidney disease.
UPLC-MS/MS Method for Simultaneous Quantification of Cyclosporine A and Urolithin A in Plasma and Interspecies Analysis Across Mammals Including Humans.
A Fully Validated LC-QTOF-MS Screening Workflow for the Analysis of Drugs in Oral Fluid.
Longitudinal urine metabolic profiling and gestational age prediction in human pregnancy.
Critical overview of mass spectrometry-based lipidomics approach for evaluating lipid oxidation in foods.
Part B: SLICE-MSI-A Machine Learning Interface for System Suitability Testing of Mass Spectrometry Imaging Platforms.
Chiral Separation and Determination of Multiple Organophosphorus Pesticide Enantiomers in Soil Based on Cellulose-Based Chiral Column by LC-MS/MS.

Biomed Chromatogr. 2025 Apr;39(4): e70023

A Sensitive and Selective LC-MS/MS-ESI Method for the Quantitation of Metabolites M9, M12, and M20 of Bexicaserin in Human Plasma and Urine Matrices.

Raja Reddy Kallem, Maisy Yeager, Rosa Chan, Katharine Fletcher, Katie Neal, Nuggehally R Srinivas.

  Bexicaserin is a highly selective 5HT2c receptor agonist being developed for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We report an LC-MS/MS method for the quantitative estimation of three pharmacologically inactive metabolites (M9, M12, and M20) of bexicaserin in human plasma/urine. Sample preparation involves the extraction of M9, M12, M20, and internal standards (ISs) from 25-μL plasma and 50-μL urine following protein precipitation. The chromatographic separation of analytes was achieved on a HSS T3-C18 column. The calibration curves ranged from 0.1 to 100 ng/mL for M9, 0.5-500 ng/mL for M12, and 1.0-1000 ng/mL for M20 in plasma and 2.0-2000 ng/mL for M9 and M12 and 10-10,000 ng/mL for M20 in urine. Intraday/interday precision and accuracy, linearity, matrix effect, extraction recovery, carry-over, dilution integrity, stability studies, and incurred sample reanalysis were performed in both plasma and urine. The intraday and interday accuracy and precision for metabolites met the stipulated regulatory guidelines. Stability studies in plasma and urine showed that analytes were stable at bench-top for > 23.5 h and in autosampler for > 69 h. Analytes were stable after five freeze-thaw cycles and > 552 days of long-term storage at -20°C and -80°C.

Keywords:  DEE; LC–MS/MS; LP352; bexicaserin; metabolites; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.70023
Biomed Chromatogr. 2025 Apr;39(4): e70030

Simultaneous Quantification of Filgotinib and Its Active Metabolite in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry: Validation and Clinical Application.

Takahiro Ito, Manabu Suno, Minae Shintani, Ayaka Iwata, Takao Fujii, Kazuo Matsubara.

  Filgotinib (FLG) is a Janus kinase 1 inhibitor and is metabolized to an active metabolite, GS-829845. There is no report on the method for simultaneous quantification of FLG and GS-829845 in clinical samples. We developed a liquid chromatography-tandem mass spectrometry method for simultaneous determination of FLG and GS-829845 in patient plasma. FLG and GS-829845 were extracted from an aliquot of 50 μL of human plasma by simple deproteinization using methanol. Chromatographic separation was performed using a Shim-pack Scepter C18-120 column with a combined mobile phase of water and methanol containing 0.1% formic acid and gradient elution at a flow rate of 0.2 mL/min. Detection was performed by positive electrospray ionization using a QTRAP 4500 mass spectrometer. The method was validated in the concentration range of 2.5-50 ng/mL for FLG and 250-5000 ng/mL for GS-829845. Intra- and inter-day assay accuracy and precision were within 11.4% and 13.9%, respectively. Recoveries and matrix effects were consistent and reproducible. This developed and fully validated method is simple, rapid, and cost-effective and was used successfully for therapeutic drug monitoring in a patient with rheumatoid arthritis.

Keywords:  GS‐829845; JAK inhibitor; LC–MS/MS; filgotinib; rheumatoid arthritis

DOI:  https://doi.org/10.1002/bmc.70030
J Sep Sci. 2025 Feb;48(2): e70099

From Complexity to Clarity: Expanding Metabolome Coverage With Innovative Analytical Strategies.

Kanukolanu Aarika, Ramijinni Rajyalakshmi, Lakshmi Vineela Nalla, Siva Nageswara Rao Gajula.

  Metabolomics, a powerful discipline within systems biology, aims at comprehensive profiling of small molecules in biological samples. The challenges of biological sample complexity are addressed through innovative sample preparation methods, including solid-phase extraction and microextraction techniques, enhancing the detection and quantification of low-abundance metabolites. Advances in chromatographic separation, particularly liquid chromatography (LC) and gas chromatography (GC), coupled with high-resolution (HR) mass spectrometry (MS), have significantly improved the sensitivity, selectivity, and throughput of metabolomic studies. Cutting-edge techniques, such as ion-mobility mass spectrometry (IM-MS) and tandem MS (MS/MS), further expand the capacity for comprehensive metabolite profiling. These advanced analytical platforms each offer unique advantages for metabolomics, with continued technological improvements driving deeper insights into metabolic pathways and biomarker discovery. By providing a detailed overview of current trends and techniques, this review aims to offer valuable insights into the future of metabolomics in human health research and its translational potential in clinical settings. Toward the end, this review also highlights the biomedical applications of metabolomics, emphasizing its role in biomarker discovery, disease diagnostics, personalized medicine, and drug development.

Keywords:  hydrophilic interaction liquid chromatography; ion‐mobility mass spectrometry; liquid chromatography–mass spectrometry (LC–MS); metabolomics; super critical fluid chromatography

DOI:  https://doi.org/10.1002/jssc.70099
bioRxiv. 2025 Feb 05. pii: 2025.02.04.636472. [Epub ahead of print]

A systematic analysis of in-source fragments in LC-MS metabolomics.

Yuanye Chi, Joshua M Mitchell, Shuzhao Li.

The "dark matter" of metabolomics refers to the large number of unidentified features in metabolomic studies, mostly from mass spectrometry (MS) based analysis (deSilva2015; David2021; Giera2024). The topic is pertinent to the analytical coverage of small molecules in biomedical research (Kind2009; Uppal2016), approaches to metabolite annotation (Domingo2018; Chaleckis2019; Metz2025), mapping reaction pathways (Zamboni2015) and the promise of applying metabolomics and exposomics to precision medicine (Wishart2016; Vermeulen2020). The number of unidentified features is not a direct account of number of compounds, as a metabolite can have isotopologues, adducts and fragments that are measured in the same data (Mahieu2017; Wang2019; Li2023a). Giera et al (2024) recently reported that in-source fragments (ISFs) accounted for over 70% of MS/MS features in METLIN, one of the leading spectral databases, suggesting that ISFs could be a significant portion of the "dark matter". Since the reference spectra in METLIN are based on chemical standards, we examine here the LC-MS (liquid chromatography coupled mass spectrometry) metabolomics from biological samples, which are the most relevant in biomedical investigations.

DOI: https://doi.org/10.1101/2025.02.04.636472
Bioinformatics. 2025 Feb 20. pii: btaf081. [Epub ahead of print]

SimMS: A GPU-Accelerated Cosine Similarity implementation for Tandem Mass Spectrometry.

Tornike Onoprishvili, Jui-Hung Yuan, Kamen Petrov, Vijay Ingalalli, Lila Khederlarian, Niklas Leuchtenmuller, Sona Chandra, Aurelien Duarte, Andreas Bender, Yoann Gloaguen.

MOTIVATION: Untargeted metabolomics involves a large-scale comparison of the fragmentation pattern of a mass spectrum against a database containing known spectra. Given the number of comparisons involved, this step can be time-consuming.
RESULTS: In this work, we present a GPU-accelerated cosine similarity implementation for Tandem Mass Spectrometry (MS), with an approximately 1000-fold speedup compared to the MatchMS reference implementation, without any loss of accuracy. This improvement enables repository-scale spectral library matching for compound identification without the need for large compute clusters. This impact extends to any spectral comparison-based methods such as molecular networking approaches and analogue search.
AVAILABILITY: All code, results, and notebooks supporting are freely available under the MIT license at https://github.com/pangeAI/simms/.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btaf081
Anal Chim Acta. 2025 Mar 22. pii: S0003-2670(25)00122-9. [Epub ahead of print]1344 343728

Comprehensive tissue homogenization and metabolite extraction for application in clinical metabolomics.

Yuntao Hao, Jeannie Horak, Zorica Stijepic, Sultan Nilay Can, Luke Tu, Julia Alexandra Wolff, Berthold Koletzko.

   BACKGROUND: Metabolomics and lipidomics analysis of various biological samples offer insights into potential mechanisms of health and disease development. Tissue samples, compared to other biological samples, are less elucidated due to challenges in sample collection and lack of standardized sample preparation protocols for reproducible tissue homogenization and broad-range metabolite extraction.
RESULTS: Pork tissue samples were homogenized with six different solvent mixtures with increasing lipophilicity, followed by metabolites extraction using methanol for polar and methyl-tert-butyl ether (MTBE) in methanol (MeOH) for highly lipophilic compounds. Metabolite profiles of supernatant and homogenate extraction for three extract volumes were compared. Solvent dependent pipette tip blockage was addressed by introduction of a prewetting correction factor for non-polar homogenization solutions and low volume tissue homogenate pipetting. Upset plots were applied for multi-dimensional metabolite extraction efficiency evaluation for 24 different sample preparation conditions. The best-performing homogenization solution was PBS; MeOH (1:1; v/v), combined with a two-step polar metabolite and lipid extraction using MeOH and 75 % MTBE in MeOH employing the tissue homogenate. The optimized experimental conditions were applied on mouse pancreas tissues, providing evidence of varying metabolic pathway activities across different anatomical regions of an organ.
SIGNIFICANCE: This study introduces a comprehensive tissue sample preparation and metabolite quantification workflow, covering highly polar to highly lipophilic metabolites using targeted high performance liquid chromatography electrospray ionization triple quadrupole-linear ion trap mass spectrometer (HPLC-ESI-QTRAP-MS/MS) for absolute quantitation of amino acids, organic acids and keto-acids, acyl-carnitines, and phospho-choline lipids.

Keywords:  HPLC-ESI-QTRAP-MS/MS; Homogenate; Lipidomics; Metabolomics; Prewetting; Tissue homogenization; Upset plot

DOI:  https://doi.org/10.1016/j.aca.2025.343728
Rapid Commun Mass Spectrom. 2025 Apr 30. 39(8): e9993

Part A: Implementing an Analyte Panel and Sampling Protocol for Quality Control in Mass Spectrometry Imaging.

Quinn Mills, Russell R Kibbe, Alexandria L Sohn, Andrew J Percy, Krista Backiel, David C Muddiman.

   RATIONALE: While quality control (QC) and system suitability testing (SST) methods are commonly employed in mass spectrometry, the field of mass spectrometry imaging (MSI) currently lacks any universally accepted QC/SST protocols. These methods can prevent the loss of precious samples due to suboptimal instrument conditions and/or data quality, but they are more challenging to implement on MSI platforms. Herein, a panel of analytes is conveniently analyzed in a setup that reflects a typical MSI imaging experiment, and guidance is provided for downstream QC/SST evaluation.
METHODS: The analyte panel will be commercially available and consists of three pairs of unlabeled (NAT) analytes and their stable isotope-labeled (SIL) analogues; a deviation from the standard procedure is also included, which incorporates a polymer to expand m/z coverage. The NAT three-plex (or four-plex with the added polymer) is analyzed as a droplet on a slide, and the SIL three-plex is doped into the electrospray solvent, isolating the NAT and SIL compounds to different source components. Datasets are collected on clean and compromised instruments to inform QC/SST software and later evaluate instrument conditions or isolated metrics of data quality.
RESULTS: A procedure was created for QC/SST analysis on MSI platforms, which can be optionally paired with the freely available software Supervised Learning for Instrument Classification and Evaluation for Mass Spectrometry Imaging (SLICE-MSI) to classify the condition of the instrument. The SIL data may be monitored separately during imaging experiments for continuous evaluation of electrospray stability. The protocol highlights areas that may be adapted for other ionization sources for widespread use.
CONCLUSIONS: The protocol described herein uses a panel of NAT and SIL compounds to offer an objective and accurate determination of QC/SST on MSI platforms.

Keywords:  IR‐MALDESI; mass spectrometry imaging; quality control; stable isotope labeled; system suitability testing

DOI:  https://doi.org/10.1002/rcm.9993
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Feb 12. pii: S1570-0232(25)00075-3. [Epub ahead of print]1255 124523

A dilute-and-shoot LC-MS/MS determination of low-dosage third-generation antipsychotics and their metabolites in urine using an ultra-short column.

Jeong Eun Kim, Seon Yeong Kim, Jae Chul Cheong, Jin Young Kim.

  Third-generation atypical antipsychotics, known for their enhanced efficacy and reduced side effects compared to previous generations, are now extensively utilized in the treatment of schizophrenia. Due to their chemical properties and low dosages, these drugs are present at low concentrations in urine, making it challenging to monitor medication compliance among probationers. In this study, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of three third-generation antipsychotics and their main metabolites in urine. A dilute-and-shoot approach was employed for rapid urine sample preparation. All compounds were separated on an ultra-short column (2.1 × 5 mm, 1.7 μm) and detected rapidly within a span of two minutes, thereby enhancing the efficiency in handling increased workloads. The limits of detection ranged from 0.01 to 0.23 ng/mL for all compounds, with correlation coefficients exceeding 0.997. The analytical method was validated using various parameters, including selectivity, precision and accuracy, matrix effect, and stability, ensuring its reliability for forensic applications. This newly developed LC-MS/MS method was successfully applied to analyze 86 urine samples obtained from probationers undergoing antipsychotic medication. Consequently, this method proves to be useful in verifying medication compliance among probationers, and effectively managing the recent increase in the number of urine drug testing.

Keywords:  Dilute and shoot LC-MS/MS; Medication compliance; Third generation atypical antipsychotics; Ultra short column; Urine

DOI:  https://doi.org/10.1016/j.jchromb.2025.124523
Anal Bioanal Chem. 2025 Feb 21.

Quantitation of antibiotics in fresh fermentation medium by hydrophilic interaction chromatography mass spectrometry.

Nadia Marcon, Mathias Rüdt, Joachim Klein, Saša M Miladinović.

  This study presents the development of a sophisticated liquid chromatography-mass spectrometry approach leveraging hydrophilic interaction chromatography (HILIC) for the quantification of kanamycin and spectinomycin in fermentation media. The method was validated per International Council for Harmonisation guidelines, demonstrating robust linearity, precision, and accuracy. To mitigate pronounced matrix effects common to complex fermentation matrices, sample preparation was thoroughly optimized with solid-phase extraction employing MCX sorbent, thereby enhancing recovery rates and minimizing analytical interference. The validated protocol demonstrated high correlation coefficients (R > 0.998), underscoring its robustness and reliability for the accurate quantification of antibiotics in challenging bioprocess environments, providing a valuable analytical tool for bioreactor system monitoring.

Keywords:  Antibiotics; Fresh fermentation medium; HILIC-MS; Matrix effect; Solid-phase extraction

DOI:  https://doi.org/10.1007/s00216-025-05775-6
Biomed Chromatogr. 2025 Apr;39(4): e70032

Quantification of Pectolinarin in Rat Plasma Using UPLC-MS/MS and Its Pharmacokinetic Analysis.

Mengmeng Shao, Runrun Wang, Congcong Wen, Xianqin Wang, Yongxi Jin, Saiya Chen.

  Pectolinarin is a flavonoid compound known for its wound-healing properties, including anti-inflammatory and antibacterial effects. In this study, we employed UPLC-MS/MS to quantify pectolinarin in rat plasma and investigate its pharmacokinetics. Plasma samples were processed using an acetonitrile precipitation method. Chromatographic separation was performed on a UPLC BEH column with a gradient mobile phase of acetonitrile-water (containing 0.1% formic acid). Detection was carried out using electrospray ionization (ESI) tandem mass spectrometry in multiple reaction monitoring (MRM) mode with positive ionization, targeting transitions of m/z 623.3 → 315.3 for pectolinarin and m/z 370.5 → 125.0 for the IS. The results demonstrated that pectolinarin exhibited acceptable linearity in rat plasma within the concentration range of 1.2 to 2300 ng/mL (r > 0.995). The intraday and interday precision, expressed as relative standard deviation (RSD), was below 9.2%. Accuracy ranged from 97.3% to 108.3%, with average recovery exceeding 94.7%. The matrix effect was between 97.8% and 105.3%. The method was successfully applied to evaluate the pharmacokinetics of pectolinarin in rats following both oral and intravenous administration. The absolute bioavailability of pectolinarin in rats was determined to be 0.28%.

Keywords:  UPLC‐MS/MS; bioavailability; pectolinarin; pharmacokinetics; rat

DOI:  https://doi.org/10.1002/bmc.70032
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Feb 10. pii: S1570-0232(25)00071-6. [Epub ahead of print]1255 124519

Salting-out assisted liquid-liquid extraction for UPLC-MS/MS determination of bile acids and kynurenine-, indole- and serotonin-pathway metabolites of tryptophan in human serum of healthy probands.

Celine Oanes, Marina Alexeeva, Kjetil Søreide, Cato Brede.

  The bacterial composition of the gut has been found to affect many diseases, including several gastrointestinal cancers. The microbiome appears central in the production of certain metabolites that enter circulation, especially those from bile acids and the essential amino acid tryptophan. The tumor-microenvironment may also produce changes in metabolites, such as those from the tryptophan-kynurenine pathway, of which several compounds may be measured in the blood. As data emerges from large scale metabolomics studies, there will be a need to validate metabolomic biomarkers to confirm their clinical utility. This task also requires knowledge about biological variation of the same metabolites in a healthy population. For this purpose, a novel method was developed for quantification of bile acids and tryptophan metabolites in samples of human serum by ultra-performance liquid chromatography coupled with tandem mass spectrometry. Salting-out assisted liquid-liquid extraction was optimized with the ion-pairing reagent trifluoroacetic acid. In this way, both polar tryptophan metabolites and non-polar bile acids could be extracted with a high recovery, favorable matrix effects, and improved chromatographic focusing, by using straightforward robot pipetting. The instrumental analysis was fast (4 min and 32 s) and with sample injections done directly from the extraction microplate. The method was applied to quantify metabolites in serum from healthy probands, and for investigating inter- and intraindividual variations over six hours.

Keywords:  Bile acids; Liquid chromatography; Mass spectrometry; Salting-out assisted liquid-liquid extraction; Tryptophan metabolites

DOI:  https://doi.org/10.1016/j.jchromb.2025.124519
J Chromatogr A. 2025 Feb 09. pii: S0021-9673(25)00106-2. [Epub ahead of print]1745 465757

In-situ formation of deep eutectic supramolecule based extraction method coupled to valve switching ion chromatography mass spectrometry for the determination of aminoglycosides in meat.

Xuxia Liang, Zhixiong Zhong, Jianchao Deng, Runkun Zhang.

  A novel extraction methodology was developed for extracting nine aminoglycosides (AGs) from meats based on the in-situ formation of deep eutectic supramolecule between analytes and gallic acid. Good chromatographic separation was achieved on a cation-exchange column utilizing solely a diluted organic acid as the eluent. A matrix-switching system was created to divert common inorganic cations from the column effluent to waste, reducing conductance peak values by 99.8 % and mitigating ion suppression in high-resolution electrospray ionization. The methodology was validated and exhibited excellent linearity across a concentration range of 20-800 μg L-1, with correlation coefficients (r) ranging from 0.9982 to 0.9997. The limits of detection and quantification were in the range of 7.1-13.8 μg kg-1 and 22.8-44.0 μg kg-1, respectively. The method was applied to the analysis of nine AGs in forty-five samples, attaining recovery values ranging from 89.1 % to 102.1 % with relative standard deviations of 1.9 %-6.4 %. The study provides a reliable procedure that complies with the requirements of the EU's official methods of analysis for identifying and quantifying the restricted and prohibited substances. The method simplifies operations and shortens extraction times by ingeniously integrating various techniques. It also eliminates the need for toxic reagents and streamlines operations compared to the conventional liquid chromatography. The results showed that IC-MS is a convenient and selective complementary approach to other modes of chromatography for determining multiple AGs in complex samples.

Keywords:  Aminoglycosides; Beef and chicken; Deep eutectic supramolecule; Ion chromatography-mass spectrometry; Pork; Solid-liquid extraction

DOI:  https://doi.org/10.1016/j.chroma.2025.465757
Anal Chem. 2025 Feb 19.

From Reverse Phase Chromatography to HILIC: Graph Transformers Power Method-Independent Machine Learning of Retention Times.

Cailum M K Stienstra, Emir Nazdrajić, W Scott Hopkins.

Liquid chromatography (LC) is a cornerstone of analytical separations, but comparing the retention times (RTs) across different LC methods is challenging because of variations in experimental parameters such as column type and solvent gradient. Nevertheless, RTs are powerful metrics in tandem mass spectrometry (MS2) that can reduce false positive rates for metabolite annotation, differentiate isobaric species, and improve peptide identification. Here, we present Graphormer-RT, a novel graph transformer that performs the first single-model method-independent prediction of RTs. We use the RepoRT data set, which contains 142,688 reverse phase (RP) RTs (from 191 methods) and 4,373 HILIC RTs (from 49 methods). Our best RP model (trained and tested on 191 methods) achieved a test set mean average error (MAE) of 29.3 ± 0.6 s, comparable performance to the state-of-the-art model which was only trained on a single LC method. Our best-performing HILIC model achieved a test MAE = 42.4 ± 2.9 s. We expect that Graphormer-RT can be used as an LC "foundation model", where transfer learning can reduce the amount of training data needed for highly accurate "specialist" models applied to method-specific RP and HILIC tasks. These frameworks could enable the machine optimization of automated LC workflows, improved filtration of candidate structures using predicted RTs, and the in silico annotation of unknown analytes in LC-MS2 measurements.

DOI: https://doi.org/10.1021/acs.analchem.4c05859
Drug Des Devel Ther. 2025 ;19 955-969

Simultaneous Determination of Multiple Acid-Suppressing Drugs by UPLC-MS/MS Method and Application for Pharmacokinetics Study.

Xiuqi Li, Shupeng Liu, Mengyang Yu, Wanlin Xi, Xiaofei Wu, Dan Liu, Aijing Liu, Hongyun Wang.

   Background: Proton pump inhibitors (PPIs) and potassium competitive acid blockers (P-CABs) are widely used to treat acid-related diseases (ARDs). Precisely quantifying their plasma levels is crucial for clinical pharmacokinetic assessments and therapeutic drug monitoring.
Aim: This study aimed to establish a generic and efficient ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the determination of five PPIs (esomeprazole, rabeprazole, ilaprazole, lansoprazole, and pantoprazole) and the P-CAB (vonoprazan) in human plasma.
Methods: The six analytes were extracted from human plasma via protein precipitation and a single dilution step. Detection was performed on a triple quadrupole tandem mass spectrometer with positive electrospray ionization. Chromatographic separation was achieved on the ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) using gradient elution. The mobile elution was composed of 0.2% formic acid in acetonitrile (mobile phase A), 0.1% ammonium hydroxide and 10 mmol/L ammonium formate in deionized water (mobile phase B). The flow rate was 0.4 mL/min, the run time was 4.5 minutes, and the injection volume was 20 µL.
Results & Conclusions: The method exhibited excellent linearity across the ranges of 0.2-200 ng/mL for PPIs and 0.5-500 ng/mL for the P-CAB. Both intra- and inter-day precision and accuracy were within the acceptance criteria, with precision ranging from 1.1% to 14.6% and accuracy ranging from 0.0% to 14.7%. Extraction recoveries were consistent, ranging from 88.1% to 96.7%, with no significant matrix effects observed. The stability of the six analytes under diverse storage and processing conditions was also confirmed, with both precision and accuracy falling within the acceptable range of 15%. The UPLC-MS/MS assay provided an efficient and reliable approach for the simultaneous determination of six acid-suppressing medications in a single analytical run. It has been successfully applied to the pharmacokinetic studies of PPIs and P-CABs, offering a valuable tool for clinical research and therapeutic drug monitoring.

Keywords:  P-CAB; PPIs; UPLC-MS/MS; acid-suppressing drugs

DOI:  https://doi.org/10.2147/DDDT.S493911
J Mass Spectrom. 2025 Mar;60(3): e5118

Comparison of Mid-Infrared and Ultraviolet Lasers Coupled to the MALDESI Source for the Detection of Secondary Metabolites and Structural Lipids in Arabidopsis thaliana.

Sarah M Ashbacher, Jeffrey G Manni, David C Muddiman.

  Matrix-assisted laser desorption electrospray ionization (MALDESI) conventionally utilizes a mid-infrared (IR) laser for the desorption of neutrals, allowing for detection of hundreds to thousands of analytes simultaneously. This platform enables mass spectrometry imaging (MSI) capabilities to not only detect specific molecules but also reveal the distribution and localization of a wide range of biomolecules across an organism. However, an IR laser comes with its disadvantages when imaging plants. At a mid-IR wavelength (2970 nm), the compartmentalized endogenous water within the leaf structure acts as an internal matrix, causing rapid heating, and, in turn, degrades the spatial resolution and signal quality. An ultraviolet (UV) laser operates at wavelengths that overlap with the absorption bands of secondary metabolites allowing them to serve as sacrificial matrix molecules. With the integration and optimization of a 355 nm UV laser into the MALDESI-MSI NextGen source for the analysis of plants, we were able to detect diverse molecular classes including flavonoids, fatty acid derivatives, galactolipids, and glucosinolates, at higher ion abundances when compared to the mid-IR laser. These results show that re-visiting UV-MALDESI-MSI, without the need for an exogenous matrix, provides a promising approach for the detection and imaging of important analytes in plants.

Keywords:  MALDESI; mass spectrometry imaging; mid‐infrared; plants; ultraviolet

DOI:  https://doi.org/10.1002/jms.5118
Metabolomics. 2025 Feb 20. 21(2): 29

Phosphorylated glycosphingolipids are commonly detected in Caenorhabditis elegans lipidomes.

Michael Witting, Liesa Salzer, Sven W Meyer, Aiko Barsch.

   INTRODUCTION: The identification of lipids is a cornerstone of lipidomics, and due to the specific characteristics of lipids, it requires dedicated analysis workflows. Identifying novel lipids and lipid species for which no reference spectra are available is tedious and often involves a lot of manual work. Integrating high-resolution mass spectrometry with enhancements from chromatographic and ion mobility separation enables the in-depth investigation of intact lipids.
OBJECTIVES: We investigated phosphorylated glycosphingolipids from the nematode Caenorhabditis elegans, a biomedical model organism, and aimed to identify different species from this class of lipids, which have been described in one particular publication only. We checked if these lipids can be detected in lipid extracts of C. elegans.
METHODS: We used UHPLC-UHR-TOF-MS and UHPLC-TIMS-TOF-MS in combination with dedicated data analysis to check for the presence of phosphorylated glycosphingolipids. Specifically, candidate features were identified in two datasets using Mass Spec Query Language (MassQL) to search fragmentation data. The additional use of retention time (RT) and collisional cross section (CCS) information allowed to filter false positive annotations.
RESULTS: As a result, we detected all previously described phosphorylated glycosphingolipids and novel species as well as their biosynthetic precursors in two different lipidomics datasets. MassQL significantly speeds up the process by saving time that would otherwise be spent on manual data investigations. In total over 20 sphingolipids could be described.
CONCLUSION: MassQL allowed us to search for phosphorylated glycosphingolipids and their potential biosynthetic precursors systematically. Using orthogonal information such as RT and CCS helped filter false positive results. With the detection in two different datasets, we demonstrate that these sphingolipids are a general part of the C. elegans lipidome.

Keywords:   Caenorhabditis elegans ; Lipid identification; Lipidomics; MassQL; Sphingolipids; Trapped ion mobility

DOI:  https://doi.org/10.1007/s11306-024-02216-w
J Bioinform Comput Biol. 2024 Dec;22(6): 2450027

Research on similarity retrieval method based on mass spectral entropy.

Li-Ping Wu, Li Yong, Xiang Cheng, Yang Zhou.

  Compound identification in small molecule research relies on comparing experimental mass spectra with mass spectral databases. However, unequal data lengths often lead to inefficient and inaccurate retrieval. Moreover, the similarity calculation methods used by commercial software have limitations. To address these issues, two mass spectrometry data processing methods namely the "splicing-filling method" and the "matching-filling method" have been proposed. In addition, an information entropy-based similarity calculation method for mass spectra is presented. The alignment method converts mass spectra of different lengths for unknown and known compounds into equal-length mass spectra, allowing more accurate calculation of similarities between mass spectra. Information entropy measurements are used to quantify the differences in intensity distributions in the aligned mass spectral data, which are then used to compare the degree of similarity between different mass spectra. The results of the example validation show that the two data alignment methods can effectively solve the problem of unequal lengths of mass spectral data in similarity calculation. The results of the mass spectral entropy method are reliable and suitable for the identification of mass spectra.

Keywords:  Small molecule compound; information entropy; mass spectrometry data; mass spectrometry entropy; similarity calculation

DOI:  https://doi.org/10.1142/S0219720024500276
J Pharm Biomed Anal. 2025 Feb 04. pii: S0731-7085(25)00070-6. [Epub ahead of print]258 116729

High throughput analysis of vancomycin in human plasma by UHPLC-MS/MS.

Xiaoqian Chen, Luyao Yu, Yingxia Guo, Jiansong You, Meiyun Shi, Yalin Xi, Lei Yin.

  An analytical assay based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) technique for absolute quantification of vancomycin in complexed biological matrix was developed in this study. Reversed phase column with gradient elution was chosen for chromatographic separation of vancomycin and internal standard (IS) norancomycin. Sample pretreatment was performed by micro-solid phase extraction (μ-SPE) with Oasis® MAX μElution Plate (I.D., 30 μm). Multiple reaction monitoring (MRM) transition was chosen for monitoring of the analytes. For vancomycin, mass-to-charge ratio (m/z) of the MRM transition was 725.3→144.1; For norvancomycin, m/z of the MRM transition was 718.3→144.2. The running time was 3 minutes for each sample. The UHPLC-MS/MS method showed a good linear relationship (R2≥0.995) in the concentration range of 0.5-100 μg/mL. The intra- and inter-day accuracies (relative error, RE) are within the range of -3.44 %-1.50 % and precisions are between 3.48 % and 10.19 %. μ-SPE could enrich the analytes and decrease the endogenous interferences, thereby improving the selectivity and sensitivity of the method. The analytical assay is selective, accurate and reproducible. The assay was successfully applied to therapeutic drug monitoring of vancomycin in clinical application.

Keywords:  Determination; Micro-solid phase extraction; Therapeutic drug monitoring; UHPLC-MS/MS; Vancomycin

DOI:  https://doi.org/10.1016/j.jpba.2025.116729
Anal Chim Acta. 2025 Mar 22. pii: S0003-2670(25)00121-7. [Epub ahead of print]1344 343727

Successive electromembrane extraction: A new insight in simultaneous extraction of polar and non-polar metabolic molecules from biological samples.

Li Chen, Yibo Yan, Changbao Hong, Xiangting Wei, Jianhua Xiong, Chuixiu Huang, Xiantao Shen.

   BACKGROUND: Simultaneous determination of different natures of analytes is of great significance for saving sample volumes and simplifying analytical procedures. However, sample preparation for the simultaneous extraction of polar and non-polar analytes represents a challenge in sample preparation. Inspired by the successive liquid-phase microextraction (sLPME) method for acidic and basic analytes that we previously developed, we first proposed an efficient successive electromembrane extraction (sEME) system by adjusting the acidity of the donor solution and using binary organic solvents for extraction of polar and non-polar targets from biological samples in this work.
RESULTS: We performed a detailed optimization of the sEME system. Here, carnitine (C0) and acylcarnitines were selected as model analytes since the demand increased especially in metabolomics studies. The combination of 2-nonanone and 2-nitrophenylpentyl ether (NPPE) was selected as supported liquid membranes (SLMs), and trichloroacetic acid (TCA) 100 % (v/v) was added to donor solution to adjust the acidity of the donor solution after the first sEME process (sEME-1). The recoveries of the targets in blood and urine were 47%-119% and 54%-118%, respectively. Moreover, the sEME systems were evaluated by liquid chromatography tandem mass spectrometry (LC-MS/MS) from biological samples. The limit of detection (LOD) and limit of quantitation (LOQ) of analytes were 0.03-1.33 ng mL-1 and 0.09-4.42 ng mL-1, respectively.
SIGNIFICANCE: sEME enabled the extraction of polar and non-polar analytes from the same sample under optimal extraction conditions for all target analytes, which provided ideas for efficient sEME of exogenous and endogenous analytes from biological samples for forensic, clinical, and epidemiological studies.

Keywords:  Binary organic solvents; Carnitine and acylcarnitines; Polar and non-polar analytes; Successive electromembrane extraction; Urine; Whole blood

DOI:  https://doi.org/10.1016/j.aca.2025.343727
ACS Pharmacol Transl Sci. 2025 Feb 14. 8(2): 557-565

Performance Comparison of Liquid Chromatography and Paper Spray Ionization with Mass Spectrometry for Measuring Kinase Inhibitors in Human Plasma.

Richard G Lahr, Makenzie Meyer, Leah Nelson, Lisa A Kottschade, Paul J Jannetto, Yifei K Yang.

Kinase inhibitors are small-molecule drugs designed to target oncogenic mutations in cancer treatment. Although less toxic than conventional chemotherapy drugs, they can cause severe adverse effects in some patients, resulting in dose reduction and cessation. To evaluate if therapeutic drug monitoring of kinase inhibitors and their metabolites can improve toxicity assessment in patients, we developed and evaluated the analytical performance of two parallel methods utilizing liquid chromatography (LC) and paper spray (PS) ionization coupled with a triple quadrupole mass spectrometer (MS) for the measurement of dabrafenib, its major metabolite OH-dabrafenib, and trametinib in patient plasma samples. The PS-MS method yielded a faster sample analysis time (2 min) compared to the LC separation (9 min). The two methods shared the same analytical measurement range (AMR) for dabrafenib and OH-dabrafenib (10-3500 and 10-1250 ng/mL), but the AMR differed for trametinib (LC-MS: 0.5-50 ng/mL; PS-MS: 5.0-50 ng/mL). The imprecision across their respective AMR was 1.3-6.5% (dabrafenib), 3.0-9.7% (OH-dabrafenib), and 1.3-5.1% (trametinib) for the LC-MS method and 3.8-6.7% (dabrafenib), 4.0-8.9% (OH-dabrafenib), and 3.2-9.9% (trametinib) for the PS-MS method. Using authentic patient samples, the quantification results were comparable between the two methods: dabrafenib (correlation coefficient r = 0.9977), OH-dabrafenib (r = 0.885), and trametinib (r = 0.9807). Nonetheless, the PS-MS method displayed significantly higher variations compared with the LC-MS method. Based on the LC-MS method, we were able to profile the concentrations and metabolism patterns of dabrafenib and trametinib in patients who were receiving the drugs for BRAF V600 mutation-driven malignancies.

DOI: https://doi.org/10.1021/acsptsci.4c00646
Talanta. 2025 Feb 14. pii: S0039-9140(25)00230-9. [Epub ahead of print]288 127740

An automated liquid-liquid extraction platform for high-throughput sample preparation of urinary phthalate metabolites in human biomonitoring.

Yangxu Luo, Rong He, Lisong Zhang, Pengzhe Qin, Zhijun Bai, Rongfei Peng, Hui He, Lei Tan.

  Automated sample preparation reduces variation caused by human factors and improves efficiency, throughput, and reliability, making it especially important in large-scale epidemiological biomonitoring applications. In this study, we demonstrated an automated liquid-liquid extraction platform that streamlines sample preparation for human biomonitoring of urinary phthalate metabolites. This platform integrates temperature-controlled enzymatic hydrolysis, adds extraction solvents, conducts shaking extraction, performs centrifugal separation, and transfers liquids. We optimized extraction solvents for liquid-liquid extraction of urinary phthalate metabolites and compared the extraction efficiency between manual and automated methods. The analytical performance of the platform was validated and compared with those obtained by manual liquid-liquid extraction and solid-phase extraction methods. We applied the automated liquid-liquid platform for determining urinary phthalate metabolites in the human biomonitoring of 232 health participants and evaluated their association with oxidative stress levels. Urinary phthalate metabolite concentrations showed a clear declining trend with increasing age. Males had significantly higher total urinary concentrations of phthalate metabolites than females. Monobutyl phthalate was the dominant metabolite in urine samples, followed by mono-isobutyl phthalate and monoethyl phthalate, with minor gender differences observed among individual metabolites. Trend tests and Bayesian Kernel Machine Regression analysis showed a significant positive association between urinary phthalate metabolites and the oxidative stress markers 8-hydroxyguanosine and 8-hydroxy-2'-deoxyguanosine, and monobutyl phthalate was identified as the most significant metabolite for the elevated 8-hydroxy-2'-deoxyguanosine levels. The automated liquid-liquid extraction platform exhibited high efficiency and reliability in preparing urinary samples for phthalate metabolite analysis, showing great promise in large-scale sample preparation of human biomonitoring applications.

Keywords:  Automated liquid-liquid extraction; Human biomonitoring; Oxidative stress; Phthalate metabolites; Sample preparation

DOI:  https://doi.org/10.1016/j.talanta.2025.127740
J Pharm Biomed Anal. 2025 Feb 12. pii: S0731-7085(25)00079-2. [Epub ahead of print]258 116738

Validation of a quantitation method for conjugated quercetin in human plasma.

Yui Sudaka, Takafumi Mitsui, Hiroaki Kida, Mst Julia Sultana, Miyu Nishikawa, Shinichi Ikushiro, Naoto Yamaguchi.

  Since the type of glycoside affects the pharmacokinetic profile of the aglycon after oral ingestion of quercetin glycosides, clinical studies on the pharmacokinetics of quercetin glycosides are required. However, a suitable method to determine the concentrations of quercetin phase II metabolites in human plasma and urine is lacking. Therefore, we developed and validated an LC-MS method for the quantitation of conjugated quercetin using relevant reference standards, including hetero-conjugates with glucuronic acid and sulfonic acid (QC-GA/S). Quercetin hetero-conjugates extracted from rat serum were used for the method development, and reference standards were biosynthesized for the quantitation. The use of a solid-phase extraction (SPE) column in a 96 well format enabled high-throughput analysis of up to 96 tests in a day, without compromising recovery and sensitivity. The SPE column with a weak anion exchange group contributed to the high recovery of QC-GA/S. The method was then validated, and its usefulness was confirmed using clinical samples. QC-GA/S was the predominant phase II quercetin metabolite after the ingestion of quercetin glucoside or quercetin supplements. Moreover, the two peaks of QC-GA/S found in human plasma and urine were isomers of QC-7GA/4'S, which has been reported as the predominant peak in rat plasma. If QC-GA/S in plasma is responsible for a physiological activity of quercetin, it is important to determine the concentration of each QC-GA/S isomer.

Keywords:  Flavonoids; LC-MS; Pharmacokinetics; Phase-II-metabolites; Quercetin; Solid-phase-extraction

DOI:  https://doi.org/10.1016/j.jpba.2025.116738
Clin Chem Lab Med. 2025 Feb 19.

Analytical performance evaluation and optimization of serum 25(OH)D LC-MS/MS measurement.

Weiyan Zhou, Meiliang Gong, Yuanli Mao, Xiaofen Yuan, Yuhang Deng, Qianwen Zhang, Wei Guo, Ling Qiu, Xianzhang Huang, Zheng Cao, Jun Xia, Xuhui She, Yulong Cong, Chuanbao Zhang, Huafen Liu, Wenxiang Chen.

   OBJECTIVES: Measuring serum 25-hydroxyvitamin D is key in clinical labs, but inter-laboratory variations risk diagnostic errors. This study evaluates the performance of current in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods used in top Chinese clinical laboratories and proposes an optimized method for improving serum 25(OH)D measurement accuracy and reliability.
METHODS: Seven serum pools with different concentrations of 25(OH)D were prepared and sent to 12 participating laboratories for multiple repeat analysis with their current in-house LC-MS/MS methods and then an optimized LC-MS/MS method. Precision was assessed in terms of coefficient of variance (CV), and trueness was assessed in terms of bias referring to the U.S. National Institute of Standards and Technology (NIST) reference measurement procedure (RMP). The analytical performances of the two methods were compared and evaluated.
RESULTS: Eighty percent and 90 % of the laboratories achieved the defined performance criteria (CV, <12.5 %; mean bias, <8.3 %) with the optimized method for the measurement of 25(OH)D2 and 25(OH)D3, compared with 43 % and 57 % of the laboratories meeting the criteria with their in-house methods, respectively. Precision and trueness improved after applying the optimized method. Although the optimized method didn't not ensure that all laboratory samples meet the measurement uncertainty (MU) criteria (MU<13.6 %), particularly for low-concentration samples, it significantly reduced the MU compared to the in-house method.
CONCLUSIONS: Precision, trueness and MU improved after applying the optimized method. Nonetheless, more efforts are needed to ensure the reliability and accuracy of 25(OH)D measurements in clinical laboratories in China.

Keywords:  25-hydroxyvitamin D2; 25-hydroxyvitamin D3; analytical performance comparison; liquid chromatography-tandem mass spectrometry (LC-MS/MS); quality control

DOI:  https://doi.org/10.1515/cclm-2024-1416
J Chromatogr A. 2025 Feb 12. pii: S0021-9673(25)00105-0. [Epub ahead of print]1746 465756

Recent advances in lipid analysis by capillary electromigration methods, 2019-2024.

Pierre Gavard, Amélie Gavard, Lucie Perquis, Fabrice Collin, François Couderc.

  Following a long period during which Capillary Electrophoresis (CE) was little used for lipid analysis (see Poinsot et al., Electrophoresis, 40, 2019, 190-211), the last five years have seen an increase in publications on this subject. Micellar Electrokinetic Chromatography (MEKC) can now compete with Gas Chromatography (GC) for the analysis of fatty acids, while non-aqueous capillary electrophoresis (NACE) now allows the study of fatty acids as well as phospholipids or glycolipids. As NACE also allows easy coupling to Mass Spectrometry (MS) in both positive and negative Electrospray Ionization (ESI), the technique has now become sufficiently robust, and for laboratories equipped with GC or Liquid Chromatography (LC) to consider using CE, particularly as it presents the advantage of much faster sample preparation than with GC for fatty acids and a resolution identical to LC for phospholipids and glycolipids. In this article, we will therefore describe the advances made in this area over the last five years.

Keywords:  Fatty acids; Lipids; Micellar kinetic chromatography; Non-aqueous capillary electrophoresis; Phospholipids

DOI:  https://doi.org/10.1016/j.chroma.2025.465756
bioRxiv. 2025 Feb 08. pii: 2025.02.05.636727. [Epub ahead of print]

Proteoform identification using multiplexed top-down mass spectra.

Zhige Wang, Xingzhao Xiong, Xiaowen Liu.

Top-down mass spectrometry (TDMS) is the method of choice for analyzing intact proteoforms, as well as their post-translational modifications and sequence variations. In TDMS experiments, multiple proteoforms are often co-fragmented in tandem mass spectrometry (MS/MS) analysis, resulting in multiplexed TD-MS/MS spectra. Since multiplexed TD-MS/MS spectra are more complex than common spectra generated from single proteoforms, these spectra pose a significant challenge for proteoform identification and quantification. Here we present TopMPI, a new computational tool specifically designed for the identification of multiplexed TD-MS/MS spectra. Experimental results demonstrate that TopMPI significantly increases proteoform identifications and reduces identification errors in multiplexed TD-MS/MS spectral analysis compared to existing tools.

DOI: https://doi.org/10.1101/2025.02.05.636727
Anal Bioanal Chem. 2025 Feb 20.

A sensitive LC-MS/MS method for the quantification of serum epoxyeicosatrienoic and dihydroxyeicosatrienoic acids in the identification of diabetic kidney disease.

Yi Liu, Anxian Huang, Yongwei Jiang, Xiaomu Kong, Meimei Zhao, Peng Gao, Ming Yang, Ziqing Kong, Wei Jia, Yongtong Cao, Liang Ma.

  Epoxyeicosatrienoic acids (EETs) are vasoactive eicosanoids with vasodilatory, anti-inflammatory, and nephroprotective properties, and metabolized to low-bioactive dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. Evidence from animal studies suggests that EETs may be potential biomarkers for diabetic kidney disease (DKD). The aim of this study was to establish a simple, sensitive and accurate LC-MS/MS method for quantifying EETs and DHETs in human serum. Samples were prepared by solid phase extraction and quantified using a "one-to-one" isotope internal standard approach. The assay required a 9-min run time per sample. EETs and DHETs demonstrated good linearity over the investigated concentration range (r2 > 0.99). The limits of quantification were 0.01 ng/mL, with accuracies ranging from 85.62 to 110.95%, intra- and inter-day variations of less than ± 15%, and matrix effects of less than ± 10%. However, 5,6-EET showed poor performance due to chemical instability and low response peaks. We successfully applied this method to rapidly analyze serum samples from 54 patients with diabetes and DKD. Levels of EETs and DHETs were downregulated in DKD patients compared to diabetic patients, and these eicosanoids showed significant negative correlations with proteinuria. This study presented a sensitive and robust LC-MS/MS method for monitoring low levels of EETs and DHETs in human serum and showed the potential for its application in the diagnosis and staging of DKD.

Keywords:  Biomarkers; Diabetic kidney disease; Dihydroxyeicosatrienoic acids; Epoxyeicosatrienoic acids; LC-MS/MS

DOI:  https://doi.org/10.1007/s00216-025-05798-z
ACS Omega. 2025 Feb 11. 10(5): 4569-4579

UPLC-MS/MS Method for Simultaneous Quantification of Cyclosporine A and Urolithin A in Plasma and Interspecies Analysis Across Mammals Including Humans.

Ingrid Marie Heyns, Raghu Ganugula, M N V Ravi Kumar, Meenakshi Arora.

In the past decade, liquid chromatography-mass spectrometry (LC-MS/MS) has become pivotal in clinical diagnosis, drug discovery, and bioanalytical science due to its high sensitivity and rapid analysis. We have developed an ultrasensitive and robust LC-MS/MS method for the simultaneous detection and quantification of cyclosporine A (CsA) and urolithin A (UA) employing ascomycin (ASC) and naringenin (NAR) as internal standards (ISTDs). The method was validated for clinical use, revealing interspecies differences between human plasma and other mammals (e.g., mouse, rat, feline, canine, and bovine serum). Validation parameters, including accuracy, precision, limits of quantification, specificity, selectivity, carryover, linearity, stability, and recovery, met acceptable ICH standards. Linear regression across the full calibration range (1-250 ng/mL for CsA and 0.5-125 ng/mL for UA) yielded an average R2 ≥ 0.999 in all mammal models. The method achieved a limit of quantification (LOQ) of 1-2.5 ng/mL across all model plasma samples with negligible carryover and demonstrated sample stability up to 96 h intra- and interday and 48 h for bovine serum. The method was successfully applied to quantify CsA and UA in canine samples following oral administration from a previous study. With a rapid run time of 6 min, this method offers high selectivity and precision, making it ideal for analyzing limited sample sizes and addressing regulatory challenges. The ability to simultaneously quantify CsA and UA has significant clinical potential for managing complex immuno-inflammatory diseases, enabling precise dose adjustments, and optimizing treatment outcomes.

DOI: https://doi.org/10.1021/acsomega.4c08515
J Anal Toxicol. 2025 Feb 17. pii: bkaf013. [Epub ahead of print]

A Fully Validated LC-QTOF-MS Screening Workflow for the Analysis of Drugs in Oral Fluid.

Cynthia Coulter, Jonah Gonzales, Campbell A Coulter, Jarrad Wagner, Christine Moore.

A simple liquid-liquid extraction procedure (LLE) followed by liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-QTOF-MS) analysis for drugs in oral fluid collected with the Quantisal™ device has been developed. The decision point cut-off concentrations were at or below those recommended by the National Safety Council's Alcohol, Drugs, and Impairment Division (NSC-ADID) for toxicological investigation of driving under the influence of drugs cases (DUID). Currently ANSI/ASB standard 120 does not cover the analysis of oral fluid collected in impaired driving investigations, instead guidance from the NSC-ADID was used. The supporting mass spectral based screening library was adapted from commercially available databases and included Tier 1 and Tier 2 recommended compounds. Further, the additional inclusion of novel psychoactive substances and synthetic cannabinoids was based on the Center for Forensic Science Research and Education's (CFSRE) quarterly publications of 2023. Metabolites from those publications were not included in this method since, with some exceptions, parent drugs are the dominant compounds in oral fluid. Briefly, Quantisal™ (1mL) was mixed with organic solvents, centrifuged, and decanted; followed by a second liquid-liquid process which extracted all the drugs in a single aliquot. A gradient liquid chromatography program using 0.1% formic acid in water and 0.1% formic acid in methanol was used and the runtime was 10 minutes. LC-QTOF-MS settings were optimized to promote greater sensitivity for a wide range of drug classes. The method was fully validated using ANSI/ASB 036 Standard Practices for Method Validation in Forensic Toxicology as guidance. Interference studies, limit of detection, precision at and around the decision points, ionization suppression/enhancement, and processed sample stability up to 96 hours were completed for each drug in the library database. While ion suppression or enhancement of the analytes varied greatly, the decision point was not significantly affected and internal standards that mimicked similar responses were chosen for each analyte. The method was applied to proficiency program samples, routine samples received into the laboratory, and blind samples screened against the search engine. The optimization of specific tune characteristics and instrument settings allowed the user to meet or exceed recommended screening limits for drugs in Quantisal™ collected oral fluid samples without the need for immunoassay testing.

DOI: https://doi.org/10.1093/jat/bkaf013
Brief Bioinform. 2024 Nov 22. pii: bbaf059. [Epub ahead of print]26(1):

Longitudinal urine metabolic profiling and gestational age prediction in human pregnancy.

Xiaotao Shen, Songjie Chen, Liang Liang, Monika Avina, Hanyah Zackriah, Laura Jelliffe-Pawlowski, Larry Rand, Michael P Snyder.

  Pregnancy is a vital period affecting both maternal and fetal health, with impacts on maternal metabolism, fetal growth, and long-term development. While the maternal metabolome undergoes significant changes during pregnancy, longitudinal shifts in maternal urine have been largely unexplored. In this study, we applied liquid chromatography-mass spectrometry-based untargeted metabolomics to analyze 346 maternal urine samples collected throughout pregnancy from 36 women with diverse backgrounds and clinical profiles. Key metabolite changes included glucocorticoids, lipids, and amino acid derivatives, indicating systematic pathway alterations. We also developed a machine learning model to accurately predict gestational age using urine metabolites, offering a non-invasive pregnancy dating method. Additionally, we demonstrated the ability of the urine metabolome to predict time-to-delivery, providing a complementary tool for prenatal care and delivery planning. This study highlights the clinical potential of urine untargeted metabolomics in obstetric care.

Keywords:  gestational age prediction; pregnancy; urine metabolomics

DOI:  https://doi.org/10.1093/bib/bbaf059
Food Sci Biotechnol. 2025 Mar;34(4): 837-849

Critical overview of mass spectrometry-based lipidomics approach for evaluating lipid oxidation in foods.

JaeYoon Kang, JuDong Yeo.

  Mass spectrometry-based lipidomics, developed through rapid advancements in instruments and techniques, provides comprehensive analyses of individual lipidomes in diverse biological systems. This contribution summarizes the limitations of classical methods for measuring lipid oxidation in foods and presents current novel technologies for evaluating lipid oxidation. Notably, this study introduces the mass spectrometry-based lipidomics approach and its utility in assessing lipid oxidation through various analytical modes, supported by numerous examples. This overview offers significant insights into the use of mass spectrometry-based lipidomics for measuring lipid oxidation in foods, proposing lipidomics analysis as a promising method to address the limitations of classical approaches.

Keywords:  LC–MS/MS; Lipid oxidation; Lipidomics

DOI:  https://doi.org/10.1007/s10068-024-01726-6
Rapid Commun Mass Spectrom. 2025 Apr 30. 39(8): e9990

Part B: SLICE-MSI-A Machine Learning Interface for System Suitability Testing of Mass Spectrometry Imaging Platforms.

Russell R Kibbe, Quinn Mills, Alexandria L Sohn, David C Muddiman.

   RATIONALE: The field of mass spectrometry imaging is currently devoid of standardized protocols or commercially available products designed for system suitability testing of MSI platforms. Machine learning is an approach that can quickly and effectively identify complex patterns in data and use them to make informed classifications, but there is a technical barrier to implementing these algorithms. Here we package the machine learning algorithms into a user-friendly interface to make community-wide implementation of this protocol possible.
METHODS: The software package is built entirely in the Python language using the PySimpleGUI library for the construction of the interface, Pandas and Numpy libraries for data formatting and manipulation, and the Scikit-Learn library for the implementation of machine learning algorithms. Training data is collected on an instrument under clean and compromised conditions that can then be used to evaluate model performance and to train models prior to interrogating unknown samples before, during, or after experiments.
RESULTS: Detailed instructions are provided for the effective use of the SLICE-MSI software package to use machine learning to evaluate instrument condition of MSI platforms. File formatting and generalizable steps are clearly described to make the implementation of this package easy for multiple labs and different MSI platform configurations.
CONCLUSIONS: In this protocol, we demonstrate SLICE-MSI, a machine learning graphical user interface for efficient and easy implementation of QC instrument classification of mass spectrometry imaging platforms.

Keywords:  graphical user interface; machine learning; mass spectrometry imaging; quality control; system suitability testing

DOI:  https://doi.org/10.1002/rcm.9990
J Sep Sci. 2025 Feb;48(2): e70100

Chiral Separation and Determination of Multiple Organophosphorus Pesticide Enantiomers in Soil Based on Cellulose-Based Chiral Column by LC-MS/MS.

Liang Li, Rendan Zhou, Huiying Xie, Gang Li, Zhiguang Xu, Meijia Liu, Wanting Gao.

  The widespread use of organophosphorus pesticides (OPs) has raised significant environmental and health concerns due to their residues in soil and potential entry into the food chain. This study introduced chiral analysis methods for four OPs-methamidophos (METHP), dipterex (DIP), malathion (MALA), and isothiophos-methyl (ISOME)-using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with cellulose-based chiral columns. Three distinct methods were established: one for METHP, another for DIP, and a third for MALA and ISOME. Key chromatographic variables, including organic mobile phases and column temperatures, were systematically optimized, achieving maximum resolutions (Rs) of 1.61 for METHP, 2.40 for DIP, 1.70 for MALA, and 2.02 for ISOME. The QuEChERS method was employed for sample pretreatment, ensuring high recoveries. All three methods demonstrated excellent linearity (R > 0.998), accuracy with recoveries ranging from 79% to 121%, precision with RSD% < 11%, and sensitivity with low limits of enantiomer detection (LODs) as low as 0.17 µg/kg for METHP, 0.087 µg/kg for DIP, 0.062 µg/kg for MALA, and 0.054 µg/kg for ISOME, representing a sensitivity improvement of 16-172 times compared to existing methods. Field soil samples from Yangzizhou District, Nanchang, China, revealed significant contamination by ISOME, with concentrations of a single enantiomer reaching up to 8343 µg/kg, while MALA exhibited varying enantiomeric ratios with depth. This study provides robust analytical tools for monitoring chiral OP residues in soil, contributing to food safety and environmental protection.

Keywords:  LC–MS/MS; cellulose‐based chiral columns; chiral separation; organophosphorus pesticides; soil

DOI:  https://doi.org/10.1002/jssc.70100