bims-metlip 2023-08-13 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–08–13
23 papers selected by
Sofia Costa, Matterworks

A targeted liquid chromatography mass spectrometry method for routine monitoring of cell culture media components for bioprocess development.
Extended Similarity Methods for Efficient Data Mining in Imaging Mass Spectrometry.
A universal surrogate matrix assay for urea measurement in clinical pharmacokinetic studies of respiratory diseases.
Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes.
A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.
malbacR: A Package for Standardized Implementation of Batch Correction Methods for Omics Data.
Development and Validation of a New UFLC-MS/MS Method for the Detection of Organophosphate Pesticide Metabolites in Urine.
PlantMetSuite: A User-Friendly Web-Based Tool for Metabolomics Analysis and Visualisation.
Non-target ROIMCR LC-MS analysis of the disruptive effects of TBT over time on the lipidomics of Daphnia magna.
Simultaneous LC-MS/MS quantification of SGLT2 inhibitors and antipyrine in medium and tissue from human ex vivo placenta perfusions.
Quality Control of Chemogenomic Library Using LC-MS.
Validated LC-MS/MS method for simultaneous quantification of KRASG12C inhibitor sotorasib and its major circulating metabolite (M24) in mouse matrices and its application in a mouse pharmacokinetic study.
Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry.
Spatial Metabolome Lipidome and Glycome from a Single brain Section.
Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry.
Miniaturization and Automation Protocol of a Urinary Organic Acid Liquid-Liquid Extraction Method on GC-MS.
LC-MS/MS separation and quantitation of ribavirin in chicken and comparison of different mass spectrometric platforms.
Machine learning encodes urine and serum metabolic patterns for autoimmune disease discrimination, classification and metabolic dysregulation analysis.
Qualitative and quantitative determination of phenols and their metabolites in urine by in-syringe solid-phase extraction and LC-MS/MS analysis for evaluation of virgin olive oil metabolism.
FreeFlux: A Python Package for Time-Efficient Isotopically Nonstationary Metabolic Flux Analysis.
Metabolomics in archaeological science: A review of their advances and present requirements.
Preclinical PK investigation of a novel IDO1/TDO dual inhibitor-SHR9146 in mouse plasma and tissues by LC-MS/MS.
Determination of licit and illicit drugs and metabolites in human sweat by liquid chromatography-tandem mass spectrometry.

J Chromatogr A. 2023 Aug 06. pii: S0021-9673(23)00506-X. [Epub ahead of print]1706 464281

A targeted liquid chromatography mass spectrometry method for routine monitoring of cell culture media components for bioprocess development.

Jared Kress, Eshani Nandita, Elliott Jones, Missa Sanou, John Higgins, Hari Kosanam.

  The analysis of cell culture media (CCM) components is critical for understanding cell growth kinetics and overall product quality during biomanufacturing. Given the diverse physical and chemical nature of CCM compounds present at a wide range of concentrations, there is an increasing demand for single-platform analytical assays with exceptional specificity and sensitivity. This study presents a targeted LC-MS/MS method for the identification and quantitation of 110 CCM analytes is presented, where target metabolites are monitored over an 20-min gradient. The analyte panel constitutes amino acids, vitamins, organic acids, nucleic acids, carbohydrates, and lipids. The method employs isotopically labeled standards to enable specific and accurate relative quantitation of CCM compounds based on physicochemical properties and retention time. Quantitation is performed on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. The method demonstrates strong linearity with an R2 of ≥0.99 with three orders of linear dynamic range and inter-day and intra-day precision with a%CV of <10% for spiked-in quality control samples. We also present three case studies to demonstrate method applicability in the bioprocessing space for developing vaccines and biologics.

Keywords:  Biopharma manufacturing; Cell culture; LC-MS; Mass spectrometry; Media analysis; Relative quantitation

DOI:  https://doi.org/10.1016/j.chroma.2023.464281
bioRxiv. 2023 Jul 30. pii: 2023.07.27.550838. [Epub ahead of print]

Extended Similarity Methods for Efficient Data Mining in Imaging Mass Spectrometry.

Nicholas R Ellin, Ramón Alain Miranda-Quintana, Boone M Prentice.

Imaging mass spectrometry is a label-free imaging modality that allows for the spatial mapping of many compounds directly in tissues. In an imaging mass spectrometry experiment, a raster of the tissue surface produces a mass spectrum at each sampled x , y position, resulting in thousands of individual mass spectra, each comprising a pixel in the resulting ion images. However, efficient analysis of imaging mass spectrometry datasets can be challenging due to the hyperspectral characteristics of the data. Each spectrum contains several thousand unique compounds at discrete m/z values that result in unique ion images, which demands robust and efficient algorithms for searching, statistical analysis, and visualization. Some traditional post-processing techniques are fundamentally ill-equipped to dissect these types of data. For example, while principal component analysis (PCA) has long served as a useful tool for mining imaging mass spectrometry datasets to identify correlated analytes and biological regions of interest, the interpretation of the PCA scores and loadings can be non-trivial. The loadings often containing negative peaks in the PCA-derived pseudo-spectra, which are difficult to ascribe to underlying tissue biology. Herein, we have utilized extended similarity indices to streamline the interpretation of imaging mass spectrometry data. This novel workflow uses PCA as a pixel-selection method to parse out the most and least correlated pixels, which are then compared using the extended similarity indices. The extended similarity indices complement PCA by removing all non-physical artifacts and streamlining the interpretation of large volumes of IMS spectra simultaneously. The linear complexity, O( N ), of these indices suggests that large imaging mass spectrometry datasets can be analyzed in a 1:1 scale of time and space with respect to the size of the input data. The extended similarity indices algorithmic workflow is exemplified here by identifying discrete biological regions of mouse brain tissue.

DOI: https://doi.org/10.1101/2023.07.27.550838
Biomed Chromatogr. 2023 Aug 06. e5713

A universal surrogate matrix assay for urea measurement in clinical pharmacokinetic studies of respiratory diseases.

Yang Tang, Michael Van Parys, Abigail Walker, Alexandra Drelick, Xiaorong Liang, Brian Dean, Liuxi Chen.

  In pharmacokinetic studies for respiratory diseases, urea is a commonly used dilution marker for volume normalization of various biological matrices, owing to the fact that urea diffuses freely throughout the body and is minimally affected by disease states. In this study, we developed a convenient liquid chromatography-tandem mass spectrometry (LC-MS/MS) surrogate matrix assay for accurate urea quantitation in plasma, serum and epithelial lining fluid. Different mass spectrometer platforms and ionization modes were compared in parallel. The LC method and mass spectrometer parameters were comprehensively optimized to reduce interferences, to smooth the baseline and to maximize the signal-to-noise ratio. Saline was selected as the surrogate matrix, and its suitability was confirmed by good parallelism and accurate quality control sample measurements. Reliable and robust assay performance was demonstrated by precision and accuracy, dilution integrity, sensitivity, recovery and stability, all of which met bioanalysis requirements to support clinical studies. The assay performance was also verified and better understood by comparing it with a colorimetric assay and to a surrogate analyte assay. The newly developed surrogate matrix assay has the potential to be further expanded for urea quantitation in numerous physiological matrices.

Keywords:  LC-MS/MS; bioanalysis; parallelism; surrogate matrix; urea

DOI:  https://doi.org/10.1002/bmc.5713
Anal Chem. 2023 Aug 08.

Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes.

Ioanna Tsiara, Amelie Riemer, Mario S P Correia, Ana Rodriguez-Mateos, Daniel Globisch.

The human body has evolved to remove xenobiotics through a multistep clearance process. Non-endogenous metabolites are converted through a series of phase I and different phase II enzymes into compounds with higher hydrophilicity. These compounds are important for diverse research fields such as toxicology, nutrition, biomarker discovery, doping control, and microbiome metabolism. One of the challenges in these research fields has been the investigation of the two major phase II modifications, sulfation and glucuronidation, and the corresponding unconjugated aglycon independently. We have now developed a new methodology utilizing an immobilized arylsulfatase and an immobilized β-glucuronidase to magnetic beads for treatment of human urine samples. The enzyme activities remained the same compared to the enzyme in solution. The separate mass spectrometric investigation of each metabolite class in a single sample was successfully applied to obtain the dietary glucuronidation and sulfation profile of 116 compounds. Our new chemical biology strategy provides a new tool for the investigation of metabolites in biological samples with the potential for broad-scale application in metabolomics, nutrition, and microbiome studies.

DOI: https://doi.org/10.1021/acs.analchem.3c02988
J Lab Physicians. 2023 Sep;15(3): 344-353

A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

Naresh Kumar Tripathy, Shravan Kumar Mishra, Gopi Nathan, Shreya Srivastava, Anshul Gupta, Raghavendra Lingaiah.

  Objectives Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX. Materials and Methods Serum protein precipitation was performed using acetonitrile-water containing 250 μM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy. Results The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 μM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 μM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel. Conclusion We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.

Keywords:  cancer; liquid chromatography; methotrexate; spectrometry; tandem mass; therapeutic drug monitoring

DOI:  https://doi.org/10.1055/s-0042-1760668
Anal Chem. 2023 Aug 08.

malbacR: A Package for Standardized Implementation of Batch Correction Methods for Omics Data.

Damon T Leach, Kelly G Stratton, Jan Irvahn, Rachel Richardson, Bobbie-Jo M Webb-Robertson, Lisa M Bramer.

Mass spectrometry is a powerful tool for identifying and analyzing biomolecules such as metabolites and lipids in complex biological samples. Liquid chromatography and gas chromatography mass spectrometry studies quite commonly involve large numbers of samples, which can require significant time for sample preparation and analyses. To accommodate such studies, the samples are commonly split into batches. Inevitably, variations in sample handling, temperature fluctuation, imprecise timing, column degradation, and other factors result in systematic errors or biases of the measured abundances between the batches. Numerous methods are available via R packages to assist with batch correction for omics data; however, since these methods were developed by different research teams, the algorithms are available in separate R packages, each with different data input and output formats. We introduce the malbacR package, which consolidates 11 common batch effect correction methods for omics data into one place so users can easily implement and compare the following: pareto scaling, power scaling, range scaling, ComBat, EigenMS, NOMIS, RUV-random, QC-RLSC, WaveICA2.0, TIGER, and SERRF. The malbacR package standardizes data input and output formats across these batch correction methods. The package works in conjunction with the pmartR package, allowing users to seamlessly include the batch effect correction in a pmartR workflow without needing any additional data manipulation.

DOI: https://doi.org/10.1021/acs.analchem.3c01289
Molecules. 2023 Aug 01. pii: 5800. [Epub ahead of print]28(15):

Development and Validation of a New UFLC-MS/MS Method for the Detection of Organophosphate Pesticide Metabolites in Urine.

Dileshwar Kumar, Sukesh Narayan Sinha, Kasturi Vasudev.

  To monitor human exposure to pesticides, experts commonly measure their metabolites in urine, particularly dialkyl phosphates (DAPs), which include diethyl phosphate (DEP), Diethyl thiophosphate (DETP), diethyl dithiophosphate (DEDTP), dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP) and dimethyl dithiophosphate (DMDTP)to monitor the metabolites of organophosphates. These DAP metabolites are a urinary biomarker for assessing pesticide exposure and potential health risks. This study presented a new screening method combining ultrafast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) to detect six DAP metabolites in human urine. The study also compared standard sample extraction methods, namely, liquid-liquid extraction (LLE); quick, easy, cheap, effective, ruggedand safe (QuEChERS); and lyophilization. After a comprehensive analysis of the methods used to extract the analytes, including recovery rate, repeatability and reproducibility, the liquid-liquid extraction (LLE) method was found to be the best. It had a high recovery rate, was easy to handle, required less sample volume and had a short extraction time. Therefore, the LLE method was chosen for further analysis. The results showed excellent performance with high recoveries between 93% and 102%, precise repeatability (RSD) between 0.62% and 5.46% and acceptable reproducibility values (RSD) between 0.80% and 11.33%. The method also had limits of detection (LOD) ranging from 0.0201 ng/mL to 0.0697 ng/mL and limits of quantification (LOQ) ranging from 0.0609 ng/mL to 0.2112 ng/mL. Furthermore, the UFLC-MS/MS method was validated based on the SANTE guidance and successfully analyzed 150 urine samples from farmers and non-farmers. This validated method proved useful for biomonitoring studies focusing on OP pesticide exposure. It offers several advantages, such as a reduced need for samples, chemicals and materials, and a shorter analysis time. The method is sensitive and selective in detecting metabolites in human urine, making it a valuable approach for the practical and efficient assessment of pesticide exposure.

Keywords:  DAP metabolites; UFLC–MS/MS; biomarker; organophosphate pesticides; urine

DOI:  https://doi.org/10.3390/molecules28155800
Plants (Basel). 2023 Aug 06. pii: 2880. [Epub ahead of print]12(15):

PlantMetSuite: A User-Friendly Web-Based Tool for Metabolomics Analysis and Visualisation.

Yu Liu, Hao-Zhuo Liu, Ding-Kang Chen, Hong-Yun Zeng, Yi-Li Chen, Nan Yao.

  The advancement of mass spectrometry technologies has revolutionised plant metabolomics research by enabling the acquisition of raw metabolomics data. However, the identification, analysis, and visualisation of these data require specialised tools. Existing solutions lack a dedicated plant-specific metabolite database and pose usability challenges. To address these limitations, we developed PlantMetSuite, a web-based tool for comprehensive metabolomics analysis and visualisation. PlantMetSuite encompasses interactive bioinformatics tools and databases specifically tailored to plant metabolomics data, facilitating upstream-to-downstream analysis in metabolomics and supporting integrative multi-omics investigations. PlantMetSuite can be accessed directly through a user's browser without the need for installation or programming skills. The tool is freely available and will undergo regular updates and expansions to incorporate additional libraries and newly published metabolomics analysis methods. The tool's significance lies in empowering researchers with an accessible and customisable platform for unlocking plant metabolomics insights.

Keywords:  bioinformatics tools; data visualisation; metabolite identification; omics data; plant metabolomics

DOI:  https://doi.org/10.3390/plants12152880
Metabolomics. 2023 08 07. 19(8): 70

Non-target ROIMCR LC-MS analysis of the disruptive effects of TBT over time on the lipidomics of Daphnia magna.

Jamile Mohammad Jafari, Josefina Casas, Carlos Barata, Hamid Abdollahi, Romà Tauler.

   INTRODUCTION: This study has investigated the temporal disruptive effects of tributyltin (TBT) on lipid homeostasis in Daphnia magna. To achieve this, the study used Liquid Chromatography-Mass Spectrometry (LC-MS) analysis to analyze biological samples of Daphnia magna treated with TBT over time. The resulting data sets were multivariate and three-way, and were modeled using bilinear and trilinear non-negative factor decomposition chemometric methods. These methods allowed for the identification of specific patterns in the data and provided insight into the effects of TBT on lipid homeostasis in Daphnia magna.
OBJECTIVES: Investigation of how are the changes in the lipid concentrations of Daphnia magna pools when they were exposed with TBT and over time using non-targeted LC-MS and advanced chemometric analysis.
METHODS: The simultaneous analysis of LC-MS data sets of Daphnia magna samples under different experimental conditions (TBT dose and time) were analyzed using the ROIMCR method, which allows the resolution of the elution and mass spectra profiles of a large number of endogenous lipids. Changes obtained in the peak areas of the elution profiles of these lipids caused by the dose of TBT treatment and the time after its exposure are analyzed by principal component analysis, multivariate curve resolution-alternative least square, two-way ANOVA and ANOVA-simultaneous component analysis.
RESULTS: 87 lipids were identified. Some of these lipids are proposed as Daphnia magna lipidomic biomarkers of the effects produced by the two considered factors (time and dose) and by their interaction. A reproducible multiplicative effect between these two factors is confirmed and the optimal approach to model this dataset resulted to be the application of the trilinear factor decomposition model.
CONCLUSION: The proposed non-targeted LC-MS lipidomics approach resulted to be a powerful tool to investigate the effects of the two factors on the Daphnia magna lipidome using chemometric methods based on bilinear and trilinear factor decomposition models, according to the type of interaction between the design factors.

Keywords:  Non-targeted lipidomics; ROIMCR; Three-way data analysis; Trilinear and bilinear modelling

DOI:  https://doi.org/10.1007/s11306-023-02030-w
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jul 24. pii: S1570-0232(23)00251-9. [Epub ahead of print]1228 123841

Simultaneous LC-MS/MS quantification of SGLT2 inhibitors and antipyrine in medium and tissue from human ex vivo placenta perfusions.

Sabrina Kuoni, Daniel Müller, Ana Paula Simões-Wüst, Regula Steiner.

  A liquid chromatography - tandem mass spectrometry (LC-MS/MS) method has been developed to simultaneously measure four sodium glucose co-transporter 2 (SGLT2) inhibitors and the transfer marker antipyrine (ANTI) in perfusion medium and placental tissue collected from ex vivo human placental perfusions. The four SGLT2 inhibitors were empagliflozin (EMPA), dapagliflozin (DAPA), ertugliflozin (ERTU) and canagliflozin (CANA). Chromatographic separation was achieved on an Uptisphere® C18 reversed phase column (50 mm × 4.6 mm × 5 µm) within 2.85 min, using a gradient elution with 10 mM ammonium formate in water (mobile phase A) and acetonitrile (mobile phase B) both with 0.1% formic acid. Analysis of ammonium adduct ions was performed on an AB SCIEX 6500+ triple quadrupole mass spectrometer using positive electrospray ionisation and scheduled multiple reaction monitoring (sMRM). The transitions were m/z 468.00 → 355.20 (EMPA), m/z 426.00 → 167.20 (DAPA), m/z 437.10 → 206.90 (ERTU), m/z 462.00 → 249.00 (CANA) and m/z 189.20 → 55.90 (ANTI). The method was validated according to the European Medicines Agency guidelines and was proven to be selective, linear within a concentration range of 1-1000 µg/L (DAPA, CANA, ANTI) and 1-500 µg/L (EMPA, ERTU), accurate, precise and free of carry-over, instabilities, recovery and matrix effect issues. This newly developed method is suitable to analyse perfusion medium and placenta tissue samples collected during ex vivo human placenta perfusions. It thereby enables quantification of transport across the placental barrier of the SGLT2 inhibitors EMPA, DAPA, ERTU and CANA as well as the transfer marker ANTI.

Keywords:  Ex vivo placenta perfusion; LC-MS/MS; Perfusion medium; SGLT2 inhibitors; Tissue homogenate

DOI:  https://doi.org/10.1016/j.jchromb.2023.123841
Methods Mol Biol. 2023 ;2706 51-58

Quality Control of Chemogenomic Library Using LC-MS.

Václav Němec, Stefan Knapp.

  In chemical biology, using compounds with incorrect identity or insufficient purity can lead to misleading biological activity data. Chemical quality control for confirmation of purity and compound identity is thus central to chemogenomics. We have established a medium-throughput LC-MS-based semi-automated quality control (QC) workflow with a minimal requirement for materials suitable for chemogenomics and other small molecule libraries. This rapid method can cover a broad chemical space of small organic compounds with diverse physicochemical properties such as polarity or lipophilicity.

Keywords:  Chemical integrity; Chemogenomic library; Identity; LC-MS; Liquid chromatography; Mass spectrometry; Purity; Qualitative analysis; Quantitative analysis

DOI:  https://doi.org/10.1007/978-1-0716-3397-7_4
J Pharm Biomed Anal. 2023 Jul 31. pii: S0731-7085(23)00381-3. [Epub ahead of print]235 115612

Validated LC-MS/MS method for simultaneous quantification of KRASG12C inhibitor sotorasib and its major circulating metabolite (M24) in mouse matrices and its application in a mouse pharmacokinetic study.

Irene A Retmana, Nancy H C Loos, Alfred H Schinkel, Jos H Beijnen, Rolf W Sparidans.

  We have successfully developed and validated a bioanalytical assay using liquid chromatography tandem mass spectrometry to simultaneously quantify the first approved KRASG12C inhibitor sotorasib and its major circulating metabolite (M24) in various mouse matrices. M24 was synthesized in-house via low-pH hydrolysis. We utilized a fast and efficient protein precipitation method in a 96-well plate format to extract both analytes from biological matrices. Erlotinib was selected as the internal standard in this assay. Gradient elution using methanol and 0.1 % formic acid in water (v/v) was applied on an Acquity UPLC BEH C18 column to separate all analytes. Sotorasib, M24, and erlotinib were detected with a triple quadrupole mass spectrometer in positive electrospray ionization in multiple reaction monitoring mode. During the validation and sample quantification, a linear calibration range was observed for both sotorasib and M24 in a range of 4 - 4000 nM and 1 - 1000 nM, respectively. The %bias and %CV (both intra- and inter-day) for all tested levels in all investigated matrices were lower than 15 % as required by the guidelines. Sotorasib had a rather short room temperature stability in mouse plasma for up to 8 h compared to M24 which was stable up to 16 h at room temperature. This method has been successfully applied to measure sotorasib and M24 in several mouse matrices from three different mouse strains. We can conclude that the plasma exposure of sotorasib in mice is limited via human CYP3A4- and mouse Cyp3a-mediated metabolism of sotorasib into M24.

Keywords:  KRAS inhibitor; LC-MS/MS; M24; Major circulating metabolite; Sotorasib

DOI:  https://doi.org/10.1016/j.jpba.2023.115612
Drug Test Anal. 2023 Aug 07.

Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry.

Luis Flores, Sabine Hargrave, Amel Clifford, Gwendolyne Alarcio, Benjamin C Moeller.

  The abuse of prohibited agents including peptides and basic small-molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes the development and validation of methodology capable of the detection of over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid-phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC-MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring following introduction to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the sub-ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.

Keywords:  doping; horse; liquid chromatography-mass spectrometry; peptides; quaternary ammonium drug

DOI:  https://doi.org/10.1002/dta.3544
bioRxiv. 2023 Jul 25. pii: 2023.07.22.550155. [Epub ahead of print]

Spatial Metabolome Lipidome and Glycome from a Single brain Section.

Harrison A Clarke, Xin Ma, Cameron J Shedlock, Terrymar Medina, Tara R Hawkinson, Lei Wu, Roberto A Ribas, Shannon Keohane, Sakthivel Ravi, Jennifer Bizon, Sara Burke, Jose Francisco Abisambra, Matthew Merritt, Boone Prentice, Craig W Vander Kooi, Matthew S Gentry, Li Chen, Ramon C Sun.

Metabolites, lipids, and glycans are fundamental biomolecules involved in complex biological systems. They are metabolically channeled through a myriad of pathways and molecular processes that define the physiology and pathology of an organism. Here, we present a blueprint for the simultaneous analysis of spatial metabolome, lipidome, and glycome from a single tissue section using mass spectrometry imaging. Complimenting an original experimental protocol, our workflow includes a computational framework called Spatial Augmented Multiomics Interface (Sami) that offers multiomics integration, high dimensionality clustering, spatial anatomical mapping with matched multiomics features, and metabolic pathway enrichment to providing unprecedented insights into the spatial distribution and interaction of these biomolecules in mammalian tissue biology.

DOI: https://doi.org/10.1101/2023.07.22.550155
PLoS One. 2023 ;18(8): e0289261

Determination of residue levels of rodenticide in rodent livers offered novel diphacinone baits by liquid chromatography-tandem mass spectrometry.

David A Goldade, Shane R Siers, Steven C Hess, Robert T Sugihara, Craig A Riekena.

A specific and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of the anticoagulant rodenticide diphacinone (DPN) in mouse and rat liver. Tissue samples were extracted with a mixture of water and acetonitrile containing ammonium hydroxide. The extracted sample was cleaned up with a combination of liquid-liquid partitioning and dispersive solid phase extraction. Chromatographic separation was achieved using a Waters X-Bridge BEH C-18 LC column (50 mm, 2.1 mm ID, 2.5 μm particle size) with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. The monitored transition for DPN was m/z 339.0 → 167.0 for quantitation and 339.0 → 172.0 and 339.0 → 116.0 for confirmation. The linear range was 0.5 to 375 ng/mL. The average precision of DPN, represented by the relative standard deviation of the observed concentrations, was 7.2% (range = 0.97% - 20.4%) and the average accuracy, represented by the relative error, was 5.8% (range = 1.06% - 14.7%). The recovery of DPN fortified at 3 different levels averaged 106% in rat liver and 101% in mouse liver. The established method was successfully used to determine DPN residue levels in Polynesian rats (Rattus exulans) and mice (Mus musculus) fed two different formulated baits containing DPN. The observed residue levels were consistent with values observed in other rodent studies. However, the amount of bait consumed was lower for the novel baits evaluated in this study.

DOI: https://doi.org/10.1371/journal.pone.0289261
Molecules. 2023 Aug 07. pii: 5927. [Epub ahead of print]28(15):

Miniaturization and Automation Protocol of a Urinary Organic Acid Liquid-Liquid Extraction Method on GC-MS.

Masauso Moses Phiri, Elmarie Davoren, Barend Christiaan Vorster.

  The aim of this study was to improve the extraction method for urinary organic acids by miniaturizing and automating the process. Currently, manual extraction methods are commonly used, which can be time-consuming and lead to variations in test results. To address these issues, we reassessed and miniaturized the in-house extraction method, reducing the number of steps and the sample-to-solvent volumes required. The evaluated miniaturized method was translated into an automated extraction procedure on a MicroLab (ML) Star (Hamilton Technologies) liquid handler. This was then validated using samples obtained from the ERNDIM External Quality Assurance program. The organic acid extraction method was successfully miniaturized and automated using the Autosampler robot. The linear range for most of the thirteen standard analytes fell between 0 to 300 mg/L in spiked synthetic urine, with low (50 mg/L), medium (100 mg/L), and high (500 mg/L) levels. The correlation coefficient (r) for most analytes was >0.99, indicating a strong relationship between the measured values. Furthermore, the automated extraction method demonstrated acceptable precision, as most organic acids had coefficients of variation (CVs) below 20%. In conclusion, the automated extraction method provided comparable or even superior results compared to the current in-house method. It has the potential to reduce solvent volumes used during extraction, increase sample throughput, and minimize variability and random errors in routine diagnostic settings.

Keywords:  automation; liquid-liquid extraction; method validation; miniaturization; organic acids

DOI:  https://doi.org/10.3390/molecules28155927
BMC Chem. 2023 Aug 07. 17(1): 96

LC-MS/MS separation and quantitation of ribavirin in chicken and comparison of different mass spectrometric platforms.

Daokun Xu, Haolun Huang, Wenyan Hu, Xinmei Liu, Jun Yang.

  A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of ribavirin in chicken. Samples was extracted with 0.1% formic acid and purified by Hypercarb cartridge prior to LC-MS/MS analysis. The eluates were evaporated to dryness, reconstituted in 1 mL 5mM ammonium acetate containing 5% acetonitrile (v/v) and 0.1% (v/v) formic acid. Chromatographic separation was performed on a Hypercarb analytical column under a gradient elution program with acetonitrile and 0.1% (v/v) formic acid in 5 mM ammonium acetate at a flow rate of 0.6 ml/min. The intraday and interday accuracy ranged from - 7.83 - 1.39%, and - 6.38 - 2.25%, with precisions between 1.34 - 3.88%% and 1.10 - 4.67%. The limits of detection (LODs) and limits of quantitation (LOQs) of ribavirin was 0.1 ng/mL and 0.5 ng/mL, respectively. The method was validated for linearity, accuracy, precision, matrix effect and stability. Application of the method confirmed 3 ribavirin positive samples out of 50 commercial chicken samples, with concentrations of ribavirin ranging from 0.9 μg/kg to 5.8 μg/kg a, respectively. Additionally, both AB Sciex 5500 and Agilent 6945B were proven to be suitable in ribavirin separation and quantification. The described method is suitable for the determination of ribavirin in chicken in analytical practice to monitor illegal addition of this kind of anti-viral drug.

Keywords:  Anti–viral drug; Chicken; LC–MS/MS; Ribavirin; Separation

DOI:  https://doi.org/10.1186/s13065-023-01010-4
Analyst. 2023 Aug 07.

Machine learning encodes urine and serum metabolic patterns for autoimmune disease discrimination, classification and metabolic dysregulation analysis.

Qiuyao Du, Xiao Wang, Junyu Chen, Yiran Wang, Wenlan Liu, Liping Wang, Huihui Liu, Lixia Jiang, Zongxiu Nie.

There is a wide variety of autoimmune diseases (ADs) with complex pathogenesis and their accurate diagnosis is difficult to achieve because of their vague symptoms. Metabolomics has been proven to be an efficient tool in the analysis of metabolic disorders to provide clues about the mechanism and diagnosis of diseases. Previous studies of the metabolomics analysis of ADs were not competent in their discrimination. Herein, a liquid chromatography tandem mass spectrometry (LC-MS) strategy combined with machine learning is proposed for the discrimination and classification of ADs. Urine and serum samples were collected from 267 subjects consisting of 127 healthy controls (HC) and 140 AD patients, including those with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), sicca syndrome (SS), ankylosing spondylitis (AS), systemic scleroderma (SSc) and connective tissue disease (CTD). Machine learning algorithms were encoded for the discrimination and classification of ADs with metabolomic patterns obtained by LC-MS, and satisfactory results were achieved. Notably, urine samples exhibited higher accuracy for disease differentiation and triage than serum samples. Apart from that, differential metabolites were selected and metabolite panels were evaluated to demonstrate their representativeness. Metabolic dysregulations were also investigated to gain more knowledge about the pathogenesis of ADs. This research provides a promising method for the application of metabolomics combined with machine learning in precision medicine.

DOI: https://doi.org/10.1039/d3an01051a
Talanta. 2023 Aug 03. pii: S0039-9140(23)00780-4. [Epub ahead of print]266(Pt 1): 125029

Qualitative and quantitative determination of phenols and their metabolites in urine by in-syringe solid-phase extraction and LC-MS/MS analysis for evaluation of virgin olive oil metabolism.

D Luque-Córdoba, C A Ledesma-Escobar, F Priego-Capote.

  To know the bioavailability of virgin olive oil (VOO) phenols and its impact on health, it is necessary to determine the levels of phenols excreted in urine. We present here a novel strategy for in-syringe solid-phase extraction and analysis of the extract by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using ammonium fluoride as ionization agent to enhance sensitivity. This approach allows avoiding additional steps such as solvent evaporation or analytes derivatization. The method can be used with a previous acid hydrolysis for quantitative determination of tyrosol and hydroxytyrosol to estimate metabolized phenols. We tested this application by analysis of a cohort of volunteers (n = 20) after a standardized intake of VOO. Additionally, the method can be used as such for metabolite profiling of phenolic derivatives in urine using LC-MS/MS in high-resolution data-independent acquisition (DIA). Information about the phenolic profile of the consumed VOO and the human metabolism is thus obtained. The proposed approach represents a simple and versatile tool for qualitative and quantitative characterization of VOO phenolic metabolism.

Keywords:  Data-independent acquisition; LC–MS/MS; Phenols; Urine; Virgin olive oil

DOI:  https://doi.org/10.1016/j.talanta.2023.125029
ACS Synth Biol. 2023 Aug 10.

FreeFlux: A Python Package for Time-Efficient Isotopically Nonstationary Metabolic Flux Analysis.

Chao Wu, Michael Guarnieri, Wei Xiong.

  13C metabolic flux analysis is a powerful tool for metabolism characterization in metabolic engineering and synthetic biology. However, the widespread adoption of this tool is hindered by limited software availability and computational efficiency. Currently, the most widely accepted 13C-flux tools, such as INCA and 13CFLUX2, are developed in a closed-source environment. While several open-source packages or software are available, they are either computationally inefficient or only suitable for flux estimation at isotopic steady state. To address the need for a time-efficient computational tool for the more complicated flux analysis at an isotopically nonstationary state, especially for understanding the single-carbon substrate metabolism, we present FreeFlux. FreeFlux is an open-source Python package that performs labeling pattern simulation and flux analysis at both isotopic steady state and transient state, enabling a more comprehensive analysis of cellular metabolism. FreeFlux provides a set of interfaces to manipulate the objects abstracted from a labeling experiment and computational process, making it easy to integrate into other programs or pipelines. The flux estimation by FreeFlux is fast and reliable, and its validity has been confirmed by comparison with results from other computational tools using both synthetic and experimental data. FreeFlux is freely available at https://github.com/Chaowu88/freeflux with a detailed online tutorial and documentation provided at https://freeflux.readthedocs.io/en/latest/index.html.

Keywords:  13C metabolic flux analysis; flux estimation; isotopic labeling; labeling pattern simulation; python package; steady state; transient state

DOI:  https://doi.org/10.1021/acssynbio.3c00265
Sci Adv. 2023 Aug 11. 9(32): eadh0485

Metabolomics in archaeological science: A review of their advances and present requirements.

Diego Badillo-Sanchez, Maria Serrano Ruber, Anna Davies-Barrett, Donald J L Jones, Martin Hansen, Sarah Inskip.

Metabolomics, the study of metabolites (small molecules of <1500 daltons), has been posited as a potential tool to explore the past in a comparable manner to other omics, e.g., genomics or proteomics. Archaeologists have used metabolomic approaches for a decade or so, mainly applied to organic residues adhering to archaeological materials. Because of advances in sensitivity, resolution, and the increased availability of different analytical platforms, combined with the low mass/volume required for analysis, metabolomics is now becoming a more feasible choice in the archaeological sector. Additional approaches, as presented by our group, show the versatility of metabolomics as a source of knowledge about the human past when using human osteoarchaeological remains. There is tremendous potential for metabolomics within archaeology, but further efforts are required to position it as a routine technique.

DOI: https://doi.org/10.1126/sciadv.adh0485
Front Oncol. 2023 ;13 1191778

Preclinical PK investigation of a novel IDO1/TDO dual inhibitor-SHR9146 in mouse plasma and tissues by LC-MS/MS.

Mei Xiao, Kan Zhong, Li Guo, Wei Li, Xiaoli Wang, Zhenjun Qiu, Taijun Hang.

   Purpose: The aim of the present study was to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of SHR9146, a novel IDO1/TDO dual inhibitor, in mouse plasma and tissues, and to apply it to investigate the preclinical plasma pharmacokinetics and tissue distribution of SHR9146 in mice.
Methods: Samples were spiked with deuterated SHR9146-d4 as an internal standard and pretreated by protein-precipitation extraction with methanol. Chromatographic separation was performed on a Venusil ABS C18 column (150 × 4.6 mm, 5 μm) by isocratic elution with 10 mM ammonium acetate buffer containing 0.1% formic acid solution and methanol as mobile phases. MS detection was conducted in positive electrospray ionization with multiple reaction monitoring at m/z 444.1/229.4 for SHR9146 and m/z 448.4/229.2 for the internal standard.
Results: The method showed good linearity in the calibration range from 0.05 to 50.0 μg/mL. Precisions (intra- and inter-run) were in the range from 0.5% to 5.1%, and accuracies (RE) were between -3.0% and 4.4% for all the concentration levels. SHR9146 was stable in all the tested bio-samples with recoveries >90%. Pharmacokinetic parameters were obtained by non-compartmental analysis. SHR9146 has a half-life of 0.713 h when IV-injected, with CL 12 mL/min/kg and Vd 0.666 L/kg. After oral dosing from 20 to 80 mg/kg, Cmax (range from 8.751 to 12.893 μg/mL) and AUC0-t (range from 15.606 to 69.971 μg·h/mL) of SHR9146 showed dose proportionality. Other post-oral pharmacokinetic parameters in plasma were as follows (n=6): Tmax 0.79 ± 0.36 h, t1/2 1.586 ± 0.853 h, CL 19.8 ± 0.9 mL/min/kg, Vd 3.427± 1.617 L/kg, and absolute bioavailability of 54.2% ± 12.6% (range from 40.2% to 64.7%). In addition, SHR9146 was found to be absorbed rapidly and distributed widely and mainly in the stomach, adrenal gland, liver, and lung.
Conclusion: The method was simple, sensitive, accurate, and specific and was successfully applied for the preclinical pharmacokinetic and tissue distribution study of SHR9146 in mice. The results showed that SHR9146 had dose-independent kinetics in mice via oral administration and was absorbed rapidly and distributed widely. The study provides a good basis for further drug development assessment.

Keywords:  IDO1/TDO inhibitor; LC-MS/MS; SHR9146; pharmacokinetics; tissue distribution

DOI:  https://doi.org/10.3389/fonc.2023.1191778
Anal Methods. 2023 Aug 07.

Determination of licit and illicit drugs and metabolites in human sweat by liquid chromatography-tandem mass spectrometry.

Margaret Sotom, Paul Bowdler, Kevin C Honeychurch.

A liquid chromatography-tandem mass spectrometry screening method in sweat was developed for the simultaneous determination of three licit drugs (nicotine, paracetamol, and caffeine); four illicit drugs (cocaine, ketamine, 25I-NBOMe and methamphetamine) and two metabolites (benzoylecgonine and cotinine). Target drugs were liberated from sweat patches with pH 5 sodium acetate buffer and further purified by solid phase extraction (SPE) utilising Strata-X-Drug B cartridges. Optimal solvent constituents for SPE organic wash and elution were 70% v/v methanol in deionised water and 5% v/v ammonium hydroxide in methanol respectively. Chromatographic separation was achieved using a superficially porous particle C18 column with gradient elution, using (A) 0.1% formic acid in water and (B) acetonitrile as mobile phase constituents. Target drugs were identified using a combination of retention time, and the ion ratios for two precursor-product ion transitions for each analyte monitored in multiple reaction monitoring (MRM) mode. The method was linear for all target drugs from 1.0-150.0 ng mL-1 with corresponding limits of quantitation of 1.0 ng mL-1. Limits of detection were found to range from 0.1-0.6 ng per patch. The method was subsequently applied to the analysis of sweat samples from five male and four female participants aged 20-25 years. Sweat was collected from two areas (right forearm and left thigh) using protected layers of gauze. All eighteen patches tested positive for at least one target analyte. The results of this study not only show a multi-substance screening method was achieved but also that sweat patches can be used to indicate an individual's drug use. Therefore, they can provide an alternative non-invasive technique for forensic applications.

DOI: https://doi.org/10.1039/d3ay00678f