bims-metlip 2023-02-26 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–02–26
25 papers selected by
Sofia Costa, Matterworks

Simultaneous measurement of kynurenine metabolites and explorative metabolomics using liquid chromatography-mass spectrometry: A novel accurate method applied to serum and plasma samples from a large healthy cohort.
Regularized adversarial learning for normalization of multi-batch untargeted metabolomics data.
Comprehensive Lipidomic Workflow for Multicohort Population Phenotyping Using Stable Isotope Dilution Targeted Liquid Chromatography-Mass Spectrometry.
Liquid chromatography and differential mobility spectrometry-data-independent mass spectrometry for comprehensive multidimensional separations in metabolomics.
Applied Clinical Tandem Mass Spectrometry-Based Quantification Methods for Lipid-Derived Biomarkers, Steroids and Cannabinoids: Fit-for-Purpose Validation Methods.
Four-dimensional trapped ion mobility spectrometry lipidomics for high throughput clinical profiling of human blood samples.
LC-MS-Based Targeted Metabolomics for FACS-Purified Rare Cells.
Simultaneous Quantification of Ivacaftor, Tezacaftor, and Elexacaftor in Cystic Fibrosis Patients' Plasma by a Novel LC-MS/MS Method.
UHPLC-MS/MS-Based Identity Confirmation of Amino Acids Involved in Response to and Side Effects from Antiseizure Medications.
Solvent effects of N,N-dimethylformamide and methanol on mass spectrometry imaging by tapping-mode scanning probe electrospray ionization.
Hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry combined with fabric phase sorptive extraction for therapeutic drug monitoring of pioglitazone, repaglinide, and nateglinide in human plasma.
Simultaneous determination of glyphosate, glufosinate, and their metabolites in honey using liquid chromatography-tandem mass spectrometry and solid-phase extraction.
MetaboDirect: an analytical pipeline for the processing of FT-ICR MS-based metabolomic data.
A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK).
Fast and Sensitive Analysis of Cefiderocol in Human Plasma Microsamples by Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry for Therapeutic Drug Monitoring.
Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.
Development of high-performance liquid chromatography tandem mass spectrometry assay for quantitation of ensartinib in human plasma and its application to a pharmacokinetics study in Chinese patients.
Aptamer-based sample purification for mass spectrometric quantification of trastuzumab in human serum.
Solid-phase microextraction of endogenous metabolites from intact tissue validated using a Biocrates standard reference method kit.
Applications of Mass Spectrometry in Dentistry.
Scalable Analysis of Untargeted LC-HRMS Data by Means of SQL Database Archiving.
Development, validation and application of a selective and sensitive LC-MS/MS method for the quantification of daptomycin in a suspension of Mammaliicoccus sciuri in Mueller-Hinton broth.
A Validated UHPLC-MS/MS Method to Quantify Eight Antibiotics in Quantitative Dried Blood Spots in Support of Pharmacokinetic Studies in Neonates.
Analysis of a Broad Range of Carbonyl Metabolites in Exhaled Breath by UHPLC-MS.
Metabolic Pathway Analysis: Advantages and Pitfalls for the Functional Interpretation of Metabolomics and Lipidomics Data.

J Pharm Biomed Anal. 2023 Feb 16. pii: S0731-7085(23)00073-0. [Epub ahead of print]227 115304

Simultaneous measurement of kynurenine metabolites and explorative metabolomics using liquid chromatography-mass spectrometry: A novel accurate method applied to serum and plasma samples from a large healthy cohort.

Peter Preben Eggertsen, Jakob Hansen, Malene Lundfold Andersen, Jørgen Feldbæk Nielsen, Rikke Katrine Jentoft Olsen, Johan Palmfeldt.

  Kynurenine metabolites are emerging as promising clinical biomarkers in several diseases, especially within psychiatry. Unfortunately, they are difficult to detect, particularly the challenging neurotoxic metabolite quinolinic acid (QUIN). The aim of this study was twofold: First, to develop a liquid chromatography-mass spectrometry method (LC-MS) for simultaneous targeted quantification of key kynurenine metabolites together with untargeted metabolomics, and second, to demonstrate the feasibility of the method by exploring serum/plasma and gender differences in 120 healthy young adults between 18 and 30 years of age. A range of analytical columns (C18 and biphenyl columns) and mobile phases (acidic and alkaline) were systematically evaluated. The optimized LC-MS method was based on a biphenyl column, a water-methanol gradient with 0.2% formic acid, and authentic isotope-labeled standards for each kynurenine metabolite. Precision and accuracy of targeted quantification of the key kynurenine metabolites tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), and QUIN were excellent, far exceeding the acceptance criteria specified by international guidelines. Median inter- and intra-day precision were < 6% in serum and plasma; the median accuracy was 2.4% in serum and 8% in plasma. Serum concentrations were ≤ 10% different from the corresponding concentrations in plasma for all kynurenine metabolites in healthy young adults. Men had higher levels (8-18%) of TRP, KYN, and KYNA than women (p ≤ 0.009), while no differences were observed for 3-HK and QUIN (p > 0.70). Incurred sample reanalysis of 10% of the samples yielded a median difference < 5% from the initial measurement, demonstrating the robustness of the method. Besides the targeted quantification of key kynurenine metabolites, our method was found to be suitable for simultaneous untargeted metabolomics analyses of hundreds of metabolites. A range of compound classes could be detected including amino acids, nucleic acids, dipeptides, antioxidants, and acylcarnitines, making explorative studies highly feasible. For example, we identified an additional kynurenine metabolite, 2-Quinolinecarboxylic acid, which was 47% higher in males than females (adjusted p-value = 0.001). In conclusion, in this study, we present a reliable and robust LC-MS method for simultaneous targeted and untargeted metabolomics ready for both research and clinical use. We show that both serum and plasma can be used for kynurenine studies, and the reported gender differences are in accordance with the literature. Future studies should consider using biphenyl-based LC-MS columns to successfully detect QUIN.

Keywords:  Biomarker; Human blood; Kynurenine metabolites; LC-MS; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.jpba.2023.115304
Bioinformatics. 2023 Feb 24. pii: btad096. [Epub ahead of print]

Regularized adversarial learning for normalization of multi-batch untargeted metabolomics data.

Andrei Dmitrenko, Michelle Reid, Nicola Zamboni.

MOTIVATION: Untargeted metabolomics by mass spectrometry is the method of choice for unbiased analysis of molecules in complex samples of biological, clinical, or environmental relevance. The exceptional versatility and sensitivity of modern high-resolution instruments allows profiling of thousands of known and unknown molecules in parallel. Inter-batch differences constitute a common and unresolved problem in untargeted metabolomics, and hinder the analysis of multi-batch studies or the intercomparison of experiments.
RESULTS: We present a new method, Regularized Adversarial Learning Preserving Similarity (RALPS), for the normalization of multi-batch untargeted metabolomics data. RALPS builds on deep adversarial learning with a three-term loss function that mitigates batch effects while preserving biological identity, spectral properties, and coefficients of variation. Using two large metabolomics datasets, we showcase the superior performance of RALPS as compared with six state-of-the-art methods for batch correction. Further, we demonstrate that RALPS scales well, is robust, deals with missing values, and can handle different experimental designs.
AVAILABILITY: https://github.com/zamboni-lab/RALPS.
SUPPLEMENTARY INFORMATION: Supplementary material is available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btad096
J Proteome Res. 2023 Feb 24.

Comprehensive Lipidomic Workflow for Multicohort Population Phenotyping Using Stable Isotope Dilution Targeted Liquid Chromatography-Mass Spectrometry.

Monique J Ryan, Alanah Grant-St James, Nathan G Lawler, Mark W Fear, Edward Raby, Fiona M Wood, Garth L Maker, Julien Wist, Elaine Holmes, Jeremy K Nicholson, Luke Whiley, Nicola Gray.

  Dysregulated lipid metabolism underpins many chronic diseases including cardiometabolic diseases. Mass spectrometry-based lipidomics is an important tool for understanding mechanisms of lipid dysfunction and is widely applied in epidemiology and clinical studies. With ever-increasing sample numbers, single batch acquisition is often unfeasible, requiring advanced methods that are accurate and robust to batch-to-batch and interday analytical variation. Herein, an optimized comprehensive targeted workflow for plasma and serum lipid quantification is presented, combining stable isotope internal standard dilution, automated sample preparation, and ultrahigh performance liquid chromatography-tandem mass spectrometry with rapid polarity switching to target 1163 lipid species spanning 20 subclasses. The resultant method is robust to common sources of analytical variation including blood collection tubes, hemolysis, freeze-thaw cycles, storage stability, analyte extraction technique, interinstrument variation, and batch-to-batch variation with 820 lipids reporting a relative standard deviation of <30% in 1048 replicate quality control plasma samples acquired across 16 independent batches (total injection count = 6142). However, sample hemolysis of ≥0.4% impacted lipid concentrations, specifically for phosphatidylethanolamines (PEs). Low interinstrument variability across two identical LC-MS systems indicated feasibility for intra/inter-lab parallelization of the assay. In summary, we have optimized a comprehensive lipidomic protocol to support rigorous analysis for large-scale, multibatch applications in precision medicine. The mass spectrometry lipidomics data have been deposited to massIVE: data set identifiers MSV000090952 and 10.25345/C5NP1WQ4S.

Keywords:  lipid profiling; lipid quantification; lipidomics; lipids; liquid chromatography-mass spectrometry; metabolic phenotyping; molecular epidemiology

DOI:  https://doi.org/10.1021/acs.jproteome.2c00682
Anal Bioanal Chem. 2023 Feb 23.

Liquid chromatography and differential mobility spectrometry-data-independent mass spectrometry for comprehensive multidimensional separations in metabolomics.

Lysi Ekmekciu, Gérard Hopfgartner.

  The benefits of combining drift time ion mobility (DTIMS) with liquid chromatography-high-resolution mass spectrometry (HRMS) have been reported for metabolomics but the use of differential time mobility spectrometry (DMS) is less obvious due to the need for rapid scanning of the DMS cell. Drift DTIMS provides additional precursor ion selectivity and collisional cross-section information but the separation resolution between analytes remains cell- and component-dependent. With DMS, the addition of 2-propanol modifier can improve the selectivity but on cost of analyte MS response. In the present work, we investigate the liquid chromatography-mass spectrometry (LC-MS) analysis of a mix of 50 analytes, representative for urine and plasma metabolites, using scanning DMS with the single modifiers cyclohexane (Ch), toluene (Tol), acetonitrile (ACN), ethanol (EtOH), and 2-propanol (IPA), and a binary modifier mixture (cyclohexane/2-propanol) with emphasis on selectivity and signal sensitivity. 1.5% IPA in the N2 stream was found to suppress the signal of 50% of the analytes which could be partially recovered with the use of IPA to 0.05% as a Ch/IPA mixture. The potential to use the separation voltage/compensation voltage/modifier (SV/CoV/Mod) feature as an additional analyte identifier for qualitative analysis is also presented and applied to a data-independent LCxDMS-SWATH-MS workflow for the analysis of endogenous metabolites and drugs of abuse in human urine samples from traffic control.

Keywords:  Differential mobility spectrometry; Human urine; Liquid chromatography–mass spectrometry; Metabolomics; SWATH-MS; Toxicology

DOI:  https://doi.org/10.1007/s00216-023-04602-0
Biomolecules. 2023 Feb 17. pii: 383. [Epub ahead of print]13(2):

Applied Clinical Tandem Mass Spectrometry-Based Quantification Methods for Lipid-Derived Biomarkers, Steroids and Cannabinoids: Fit-for-Purpose Validation Methods.

Isabelle Matias, Ilaria Belluomo, Pierre-Louis Raux, Monique Vallée.

  The emergence of metabolomics and quantification approaches is revealing new biomarkers applied to drug discovery. In this context, tandem mass spectrometry is the method of choice, requiring a specific validation process for preclinical and clinical applications. Research on the two classes of lipid mediators, steroids and cannabinoids, has revealed a potential interaction in cannabis addiction and metabolism-related disorders. Here we present the development of GC-MS/MS and LC-MS/MS methods for routine quantification of targeted steroids and cannabinoids, respectively. The methods were developed using an isotopic approach, including validation for linearity, selectivity, LLOQ determination, matrix effect, carryover, between- and within-run accuracy and precision, and stability tests to measure 11 steroids and seven cannabinoids in human plasma. These methods were satisfactory for most validity conditions, although not all met the acceptance criteria for all analytes. A comparison of calibration curves in biological and surrogate matrices and in methanol showed that the latter condition was more applicable for our quantification of endogenous compounds. In conclusion, the validation of our methods met the criteria for GLP-qualified rather than GLP-validated methods, which can be used for routine analytical studies for dedicated preclinical and clinical purposes, by combining appropriate system suitability testing, including quality controls in the biological matrix.

Keywords:  GC-MS/MS; LC-MS/MS; cannabinoids; human plasma; quantification methods; steroids; tandem mass spectrometry; validation

DOI:  https://doi.org/10.3390/biom13020383
Nat Commun. 2023 Feb 20. 14(1): 937

Four-dimensional trapped ion mobility spectrometry lipidomics for high throughput clinical profiling of human blood samples.

Raissa Lerner, Dhanwin Baker, Claudia Schwitter, Sarah Neuhaus, Tony Hauptmann, Julia M Post, Stefan Kramer, Laura Bindila.

Lipidomics encompassing automated lipid extraction, a four-dimensional (4D) feature selection strategy for confident lipid annotation as well as reproducible and cross-validated quantification can expedite clinical profiling. Here, we determine 4D descriptors (mass to charge, retention time, collision cross section, and fragmentation spectra) of 200 lipid standards and 493 lipids from reference plasma via trapped ion mobility mass spectrometry to enable the implementation of stringent criteria for lipid annotation. We use 4D lipidomics to confidently annotate 370 lipids in reference plasma samples and 364 lipids in serum samples, and reproducibly quantify 359 lipids using level-3 internal standards. We show the utility of our 4D lipidomics workflow for high-throughput applications by reliable profiling of intra-individual lipidome phenotypes in plasma, serum, whole blood, venous and finger-prick dried blood spots.

DOI: https://doi.org/10.1038/s41467-023-36520-1
Anal Chem. 2023 Feb 22.

LC-MS-Based Targeted Metabolomics for FACS-Purified Rare Cells.

Katharina Schönberger, Michael Mitterer, Katharina Glaser, Manuel Stecher, Sebastian Hobitz, Dominik Schain-Zota, Konrad Schuldes, Tim Lämmermann, Angelika S Rambold, Nina Cabezas-Wallscheid, Joerg M Buescher.

Metabolism plays a fundamental role in regulating cellular functions and fate decisions. Liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomic approaches provide high-resolution insights into the metabolic state of a cell. However, the typical sample size is in the order of 105-107 cells and thus not compatible with rare cell populations, especially in the case of a prior flow cytometry-based purification step. Here, we present a comprehensively optimized protocol for targeted metabolomics on rare cell types, such as hematopoietic stem cells and mast cells. Only 5000 cells per sample are required to detect up to 80 metabolites above background. The use of regular-flow liquid chromatography allows for robust data acquisition, and the omission of drying or chemical derivatization avoids potential sources of error. Cell-type-specific differences are preserved while the addition of internal standards, generation of relevant background control samples, and targeted metabolite with quantifiers and qualifiers ensure high data quality. This protocol could help numerous studies to gain thorough insights into cellular metabolic profiles and simultaneously reduce the number of laboratory animals and the time-consuming and costly experiments associated with rare cell-type purification.

DOI: https://doi.org/10.1021/acs.analchem.2c04396
Biomedicines. 2023 Feb 20. pii: 628. [Epub ahead of print]11(2):

Simultaneous Quantification of Ivacaftor, Tezacaftor, and Elexacaftor in Cystic Fibrosis Patients' Plasma by a Novel LC-MS/MS Method.

Federica Pigliasco, Alessia Cafaro, Manuela Stella, Giammarco Baiardi, Sebastiano Barco, Nicoletta Pedemonte, Claudia D'Orsi, Federico Cresta, Rosaria Casciaro, Carlo Castellani, Maria Grazia Calevo, Francesca Mattioli, Giuliana Cangemi.

  The new breakthrough cystic fibrosis (CF) drug combination of ivacaftor (IVA), tezacaftor (TEZ), and elexacaftor (ELX), namely "caftor" drugs, directly modulates the activity and trafficking of the defective CF transmembrane conductance regulator protein (CFTR) underlying the CF disease. The role of therapeutic drug monitoring (TDM) of caftor drugs in clinical settings has recently been established. The availability of reliable and robust analytical methods for the quantification of IVA, TEZ, and ELX is essential to support dose-concentration-effect studies. We have developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the rapid and simultaneous quantification of IVA, TEZ, and ELX from the plasma of CF patients. The method was based on a rapid extraction protocol from 50 μL human plasma and separation on a reversed-phase C-18 HPLC column after the addition of deuterated internal standards. Accurate analyte quantification using multiple reaction monitoring (MRM) detection was then obtained using a Thermofisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. The method has been validated following international (EMA) guidelines for bioanalytical method validation and has been tested on plasma samples from 62 CF patients treated with the three-drug combination IVA/TEZ/ELX, marketed as Kaftrio® or Trikafta®, in steady-state condition. The assay was linear over wide concentration ranges (0.008-12 mg/L) in plasma for IVA, TEZ, and ELX, suitable for a broad range of plasma concentrations, and accurate and reproducible in the absence of matrix effects. The stability of analytes for at least 30 days at room temperature could allow for cost-effective shipment and storage. On the same day of sample collection, a sweat test was evaluated for 26 associated patients' samples, FEV1 (%) for 58, and BMI was calculated for 62. However, Spearman correlation showed no correlation between Cthrough plasma concentrations of analytes (IVA, TEZ, ELX) and sweat test, FEV1 (%), or BMI. Our method proved to be suitable for TDM and could be helpful in assessing dose-concentration-response correlations in larger studies.

Keywords:  CFTR; LC–MS/MS; elexacaftor; ivacaftor; tezacaftor; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/biomedicines11020628
J Proteome Res. 2023 Feb 22.

UHPLC-MS/MS-Based Identity Confirmation of Amino Acids Involved in Response to and Side Effects from Antiseizure Medications.

Mo Awchi, Pablo Sinues, Alexandre N Datta, Diego García-Gómez, Kapil Dev Singh.

  Real-time breath analysis using secondary electrospray ionization coupled with high-resolution mass spectrometry is a fast and noninvasive method to access the metabolic state of a person. However, it lacks the ability to unequivocally assign mass spectral features to compounds due to the absence of chromatographic separation. This can be overcomed by using exhaled breath condensate and conventional liquid chromatography-mass spectrometry (LC-MS) systems. In this study, to the best of our knowledge, we confirm for the first time the presence of six amino acids (GABA, Oxo-Pro, Asp, Gln, Glu, and Tyr) previously reported to be involved in response to and side effects from antiseizure medications in exhaled breath condensate and by extension in exhaled human breath. Raw data are publicly available at MetaboLights with the accession number MTBLS6760.

Keywords:  UHPLC; amino acids; compound identification; exhaled breath condensate; tandem mass spectrometry

DOI:  https://doi.org/10.1021/acs.jproteome.2c00835
Analyst. 2023 Feb 22.

Solvent effects of N,N-dimethylformamide and methanol on mass spectrometry imaging by tapping-mode scanning probe electrospray ionization.

Yoichi Otsuka, Nijiho Ote, Mengze Sun, Shuichi Shimma, Osamu Urakawa, Shinichi Yamaguchi, Tomoya Kudo, Michisato Toyoda.

Mass spectrometry imaging (MSI) is an effective technique for visualizing the distribution of lipids in tissues. The direct extraction-ionization methods using minute volumes of solvent for local components have the advantage of rapid measurement without any sample pretreatment. For effective MSI of tissues, it is necessary to understand the effect of solvent physicochemical properties on ion images. In this study, we report solvent effects on the lipid imaging of mouse brain tissue by tapping-mode scanning probe electrospray ionization (t-SPESI) which is capable of extraction-ionization using sub-pL solvents. To precisely measure lipid ions, we developed a measurement system incorporating a quadrupole-time-of-flight mass spectrometer. The differences in signal intensity and spatial resolution of lipid ion images were investigated using N,N-dimethylformamide (non-protic polar solvent), methanol (protic polar solvent) and their mixture. The mixed solvent was suitable for the protonation of lipids, and it provided high spatial resolution MSI. Results indicate that the mixed solvent improves the extractant transfer efficiency and minimizes charged droplets from an electrospray. The solvent selectivity study revealed the importance of solvent selection based on physicochemical properties for the advancement of MSI by t-SPESI.

DOI: https://doi.org/10.1039/d2an01953a
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Feb 15. pii: S1570-0232(23)00038-7. [Epub ahead of print]1217 123628

Hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry combined with fabric phase sorptive extraction for therapeutic drug monitoring of pioglitazone, repaglinide, and nateglinide in human plasma.

Panagiotis Stamou, Anthi Parla, Abuzar Kabir, Kenneth G Furton, Dimitra Gennimata, Victoria Samanidou, Irene Panderi.

  Polypharmacy in type 2 diabetes is an issue of major concern as the prescription of multiple medi-cations for the management of diabetes-associated comorbidities can lead to drug-to-drug interactions, which can pose serious risks to patients' health. Within this context, the development of bioanalytical methods for monitoring the therapeutic levels of antidiabetic drugs is notably useful to ensure patients' safety. In the present work, a liquid chromatography-mass spectrometry method for the quantitation of pioglitazone, repaglinide, and nateglinide in human plasma is described. Sample preparation was performed by fabric phase sorptive extraction (FPSE), and hydrophilic interaction liquid chromatography (HILIC) was implemented for the chromatographic separation of the analytes, using a ZIC®-cHILIC analytical column (150 × 2.1 mm, 3 µm) under isocratic elution. The mobile phase consisted of 10 mM ammonium formate aqueous solution (pH = 6.5)/ acetonitrile, 10/90 v/v, and was pumped at a flow rate of 0.2 mL min-1. Design of Experiments was used during the development of the sample preparation method to gain deeper insight into the effect of various experimental parameters on extraction efficiency, their potential interactions and to optimize the recovery rates of the analytes. The linearity of the assay was assessed over the ranges of 25 to 2000, 6.25 to 500, and 125 to 10000 ng mL-1 for pioglitazone, repaglinide, and nateglinide, respectively. The presented method was fully validated and can be used for the therapeutic monitoring of the targeted analytes in human plasma samples.

Keywords:  Design of experiments; Fabric phase sorptive extraction; Hydrophilic interaction liquid chromatography; Hypoglycemics; Mass spectrometry

DOI:  https://doi.org/10.1016/j.jchromb.2023.123628
Anal Sci. 2023 Feb 22.

Simultaneous determination of glyphosate, glufosinate, and their metabolites in honey using liquid chromatography-tandem mass spectrometry and solid-phase extraction.

Ryoichi Sasano, Rie Ito, Masahiro Kusumoto, Junpei Sekizawa, Hiroshi Akiyama.

  Here, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of residual glyphosate, glufosinate, and their metabolites N-acetylglyphosate (Gly-A), 3-methylphosphinicopropionic acid (MPPA) and N-acetylglufosinate (Glu-A) in honey using a mixed mode column of reversed-phase and anion exchange without derivatization. The target analytes were extracted from honey samples using water, cleaned up on a reverse phase C18 cartridge column and an anion exchange NH2 cartridge column, and quantified using LC-MS/MS. Glyphosate, Glu-A, Gly-A, and MPPA were detected in negative ion mode based on deprotonation, whereas glufosinate was detected in positive ion mode. The coefficients of determination (R2) of the calibration curve, calculated in the range of 1-20 µg/kg for glufosinate, Glu-A, and MPPA, and 5-100 µg/kg for glyphosate and Gly-A, were higher than 0.993. The developed method was evaluated using honey samples spiked with glyphosate and Gly-A at 25 µg/kg and glufosinate, and MPPA and Glu-A at 5 µg/kg, based on the maximum residue levels. The validation results show good recoveries (86-106%) and precision (< 10%) for all target compounds. The limit of quantification of the developed method is 5 µg/kg for glyphosate, 2 µg/kg for Gly-A, and 1 µg/kg for glufosinate, MPPA and Glu-A. These results suggest that the developed method is applicable for quantifying residual glyphosate, glufosinate, and their metabolites in honey in compliance with Japanese maximum residue levels. Moreover, the proposed method was applied to the analysis of honey samples and glyphosate, glufosinate, and Glu-A were detected in some samples. The proposed method will be a useful tool for the regulatory monitoring of residual glyphosate, glufosinate, and their metabolites in honey.

Keywords:  Glufosinate; Glyphosate; Honey; LC–MS/MS; Residual pesticides

DOI:  https://doi.org/10.1007/s44211-023-00288-7
Microbiome. 2023 Feb 17. 11(1): 28

MetaboDirect: an analytical pipeline for the processing of FT-ICR MS-based metabolomic data.

Christian Ayala-Ortiz, Nathalia Graf-Grachet, Viviana Freire-Zapata, Jane Fudyma, Gina Hildebrand, Roya AminiTabrizi, Cristina Howard-Varona, Yuri E Corilo, Nancy Hess, Melissa B Duhaime, Matthew B Sullivan, Malak M Tfaily.

   BACKGROUND: Microbiomes are now recognized as the main drivers of ecosystem function ranging from the oceans and soils to humans and bioreactors. However, a grand challenge in microbiome science is to characterize and quantify the chemical currencies of organic matter (i.e., metabolites) that microbes respond to and alter. Critical to this has been the development of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which has drastically increased molecular characterization of complex organic matter samples, but challenges users with hundreds of millions of data points where readily available, user-friendly, and customizable software tools are lacking.
RESULTS: Here, we build on years of analytical experience with diverse sample types to develop MetaboDirect, an open-source, command-line-based pipeline for the analysis (e.g., chemodiversity analysis, multivariate statistics), visualization (e.g., Van Krevelen diagrams, elemental and molecular class composition plots), and presentation of direct injection high-resolution FT-ICR MS data sets after molecular formula assignment has been performed. When compared to other available FT-ICR MS software, MetaboDirect is superior in that it requires a single line of code to launch a fully automated framework for the generation and visualization of a wide range of plots, with minimal coding experience required. Among the tools evaluated, MetaboDirect is also uniquely able to automatically generate biochemical transformation networks (ab initio) based on mass differences (mass difference network-based approach) that provide an experimental assessment of metabolite connections within a given sample or a complex metabolic system, thereby providing important information about the nature of the samples and the set of microbial reactions or pathways that gave rise to them. Finally, for more experienced users, MetaboDirect allows users to customize plots, outputs, and analyses.
CONCLUSION: Application of MetaboDirect to FT-ICR MS-based metabolomic data sets from a marine phage-bacterial infection experiment and a Sphagnum leachate microbiome incubation experiment showcase the exploration capabilities of the pipeline that will enable the research community to evaluate and interpret their data in greater depth and in less time. It will further advance our knowledge of how microbial communities influence and are influenced by the chemical makeup of the surrounding system. The source code and User's guide of MetaboDirect are freely available through ( https://github.com/Coayala/MetaboDirect ) and ( https://metabodirect.readthedocs.io/en/latest/ ), respectively. Video Abstract.

Keywords:  Biochemical networks; FT-ICR MS; Metabolites; Organic matter

DOI:  https://doi.org/10.1186/s40168-023-01476-3
Nat Protoc. 2023 Feb 24.

A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK).

UK Consortium on Metabolic Phenotyping (MAP/UK)

Targeted metabolite assays that measure tens or hundreds of pre-selected metabolites, typically using liquid chromatography-mass spectrometry, are increasingly being developed and applied to metabolic phenotyping studies. These are used both as standalone phenotyping methods and for the validation of putative metabolic biomarkers obtained from untargeted metabolomics studies. However, there are no widely accepted standards in the scientific community for ensuring reliability of the development and validation of targeted metabolite assays (referred to here as 'targeted metabolomics'). Most current practices attempt to adopt, with modifications, the strict guidance provided by drug regulatory authorities for analytical methods designed largely for measuring drugs and other xenobiotic analytes. Here, the regulatory guidance provided by the European Medicines Agency, US Food and Drug Administration and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use are summarized. In this Perspective, we have adapted these guidelines and propose a less onerous 'tiered' approach to evaluate the reliability of a wide range of metabolomics analyses, addressing the need for community-accepted, harmonized guidelines for tiers other than full validation. This 'fit-for-purpose' tiered approach comprises four levels-discovery, screening, qualification and validation-and is discussed in the context of a range of targeted and untargeted metabolomics assays. Issues arising with targeted multiplexed metabolomics assays, and how these might be addressed, are considered. Furthermore, guidance is provided to assist the community with selecting the appropriate degree of reliability for a series of well-defined applications of metabolomics.

DOI: https://doi.org/10.1038/s41596-022-00801-8
Antibiotics (Basel). 2023 Jan 19. pii: 213. [Epub ahead of print]12(2):

Fast and Sensitive Analysis of Cefiderocol in Human Plasma Microsamples by Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry for Therapeutic Drug Monitoring.

Rossella Barone, Matteo Conti, Pier Giorgio Cojutti, Milo Gatti, Pierluigi Viale, Federico Pea.

  Cefiderocol (C) is a parenteral siderophore cephalosporin with relevant inter-individual pharmacokinetic variability among critically ill patients, which may potentially affect effective drug exposure. Therapeutic drug monitoring (TDM) may concur in improving the real-time management of C therapy in clinics. In this study, we developed and validated a fast and sensitive Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry (LC-ITD-MS/MS) method for measuring C in human plasma microsamples, as small as 3 microliters. Analysis was preceded by a user-friendly pre-analytical single-step and was performed by means of a very fast chromatographic run of 4 min, followed by positive electrospray ionization and detection on a high sensitivity triple quadrupole tandem mass spectrometer operated in multiple reaction monitoring mode. The straightforward analytical procedure was successfully validated, based on the European Medicines Agency (EMA) guidelines, in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability. The novel method was successfully applied to TDM of C in more than 50 cases of critically carbapenem-resistant Gram-negative bacterial infections and enabled us to optimize antibiotic therapy.

Keywords:  Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry; cefiderocol; plasma microsamples; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/antibiotics12020213
Methods Mol Biol. 2023 ;2615 267-280

Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.

Sushma Sharma, Ziqing Kong, Shaodong Jia, Phong Tran, Anna Karin Nilsson, Andrei Chabes.

  Defects in deoxyribonucleoside triphosphate (dNTP) metabolism are associated with a number of mitochondrial DNA (mtDNA) depletion syndromes (MDS). These disorders affect the muscles, liver, and brain, and the concentrations of dNTPs in these tissues are already normally low and are, therefore, difficult to measure. Thus, information about the concentrations of dNTPs in tissues of healthy animals and animals with MDS are important for mechanistic studies of mtDNA replication, analysis of disease progression, and the development of therapeutic interventions. Here, we present a sensitive method for the simultaneous analysis of all four dNTPs as well as all four ribonucleoside triphosphates (NTPs) in mouse muscles using hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry. The simultaneous detection of NTPs allows them to be used as internal standards for the normalization of dNTP concentrations. The method can be applied for measuring dNTP and NTP pools in other tissues and organisms.

Keywords:  Deoxyribonucleoside triphosphates; Differentiated tissues; Liquid chromatography; Triple quadrupole mass spectrometry; ZIC–HILIC

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_19
Biomed Chromatogr. 2023 Feb 24. e5610

Development of high-performance liquid chromatography tandem mass spectrometry assay for quantitation of ensartinib in human plasma and its application to a pharmacokinetics study in Chinese patients.

Hua Li, Yang Wang, Xuefei Chen, Chen Chen, Jianxin Cui, Ying Han, Lieming Ding.

  Ensartinib is a novel ALK inhibitor with potent activity against a broad range of known crizotinib-resistant ALK mutations and is developed to treat patients with non-small cell lung cancer (NSCLC). In this study, a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of ensartinib in human plasma for the first time. The plasma samples were extracted by liquid extraction and the chromatographic separation was performed on a phenomenex, Luna phenyl-hexyl (50 × 2.0 mm, 5 μm). Electrospray ionization (ESI) in positive ion mode and multiple reaction monitoring (MRM) were used to monitor ion transitions at m/z 561.3 → 257.1 (ensartinib) and 565.2 → 261.2 (IS: X-396-d4) respectively. The method appeared excellent linearity in the range of 0.5-500 ng/mL with the lowest of quantification 0.5 ng/mL. Both intra and inter-run precisions (RSD%) were less than 15% with the accuracy (RE%) between ±15%. The Extraction recovery, matrix effect, selectivity, stability were also validated and all satisfactory. Finally, the validated method was successfully applied in a phase I clinical study of ensartinib in Chinese subjects with advanced ALK-positive NSCLC.

Keywords:  Ensartinib; HPLC-MS/MS; human plasma; method validation; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.5610
Talanta. 2023 Feb 17. pii: S0039-9140(23)00100-5. [Epub ahead of print]257 124349

Aptamer-based sample purification for mass spectrometric quantification of trastuzumab in human serum.

Bo Sun, Jiuyang Liu, Pei Cai, Jianhua Wu, Wei Liu, Hankun Hu, Liang Liu.

  In this study, we developed a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify trastuzumab in human serum using aptamers for sample purification. Trastuzumab was extracted from serum samples using the capture probe based on its aptamer CH1S-3, followed by reduction, alkylation, trypsin digestion, and quantification using LC-MS/MS. Additionally, a unique peptide, FTISADTSK, was employed as a surrogate peptide and quantified, and *FTISADTSK (13C915N-labeled phenylalanine) was used as an internal standard to minimize variability in detection among the samples. The detection range for this method was 0.5-250 μg/mL, with a high correlation coefficient (r2 > 0.99). The intra- and inter-day precision (%CV, the coefficient of variation) of the quality control samples was less than 12.7%, and the accuracy (%bias) was below 8.64%. After optimization and verification, this assay was used to determine trastuzumab levels in clinical human serum samples. The results indicated that the trastuzumab concentrations had an approximate 4-fold difference among ten patients (range: 11.80-41.90 μg/mL). This study provides a novel approach for the accurate and quantitative monitoring of the mAb-trastuzumab.

Keywords:  Aptamer; Mass spectrometry; Quantification; Sample purification; Therapeutic monoclonal antibody

DOI:  https://doi.org/10.1016/j.talanta.2023.124349
J Pharm Anal. 2023 Jan;13(1): 55-62

Solid-phase microextraction of endogenous metabolites from intact tissue validated using a Biocrates standard reference method kit.

Runshan Will Jiang, Karol Jaroch, Janusz Pawliszyn.

  Improved analytical methods for the metabolomic profiling of tissue samples are constantly needed. Currently, conventional sample preparation methods often involve tissue biopsy and/or homogenization, which disrupts the endogenous metabolome. In this study, solid-phase microextraction (SPME) fibers were used to monitor changes in endogenous compounds in homogenized and intact ovine lung tissue. Following SPME, a Biocrates AbsoluteIDQ assay was applied to make a downstream targeted metabolomics analysis and confirm the advantages of in vivo SPME metabolomics. The AbsoluteIDQ kit enabled the targeted analysis of over 100 metabolites via solid-liquid extraction and SPME. Statistical analysis revealed significant differences between conventional liquid extractions from homogenized tissue and SPME results for both homogenized and intact tissue samples. In addition, principal component analysis revealed separated clustering among all the three sample groups, indicating changes in the metabolome due to tissue homogenization and the chosen sample preparation method. Furthermore, clear differences in free metabolites were observed when extractions were performed on the intact and homogenized tissue using identical SPME procedures. Specifically, a direct comparison showed that 47 statistically distinct metabolites were detected between the homogenized and intact lung tissue samples (P < 0.05) using mixed-mode SPME fibers. These changes were probably due to the disruptive homogenization of the tissue. This study's findings highlight both the importance of sample preparation in tissue-based metabolomics studies and SPME's unique ability to perform minimally invasive extractions without tissue biopsy or homogenization while providing broad metabolite coverage.

Keywords:  In vivo sampling; Metabolomics; Sample preparation; Solid-phase microextraction; Solvent extraction

DOI:  https://doi.org/10.1016/j.jpha.2022.09.002
Biomedicines. 2023 Jan 19. pii: 286. [Epub ahead of print]11(2):

Applications of Mass Spectrometry in Dentistry.

Meletia Kallianta, Eftychia Pappa, Heleni Vastardis, Christos Rahiotis.

  Mass Spectrometry (MS) is one of the fastest-developing methods in analytical instrumentation. As a highly sensitive, universal detector, it can identify known and unknown compounds, which can indeed be found in a minimal concentration. This review aims to highlight the significant milestones in MS applications in dentistry during recent decades. MS can be applied in three different fields of dentistry: (1) in research of dental materials and chemical agents, (2) in laboratory analysis of biospecimens, and (3) as a real-time diagnostic tool in service of oral surgery and pathology. MS applications on materials and agents may focus on numerous aspects, such as their clinical behavior, possible toxicity, or antimicrobial properties. MS is also a valuable, non-invasive tool for biomarkers' detection in saliva and has found great application in -omics technologies as it achieves efficient structure-finding in metabolites. As metabolites are located beyond the central dogma, this technique can provide a complete understanding of cellular functions. Thus, it is possible to determine the biological profile in normal and pathological conditions, detect various oral or systematic diseases and conditions, and predict their course. Lastly, some promising advances concerning the surgical approach to potentially oral malignant or malignant disorders exist. This breakthrough method provides a comprehensive approach to dental materials research and biomarker discovery in dental and craniofacial tissues. The current availability of various 'OMIC' approaches paves the way for individualized dentistry and provides suggestions for clinical applications in the point-of-care hubs.

Keywords:  ambient ionization mass spectrometry; mass spectrometry; proteomics; salivaomics

DOI:  https://doi.org/10.3390/biomedicines11020286
Anal Chem. 2023 Feb 20.

Scalable Analysis of Untargeted LC-HRMS Data by Means of SQL Database Archiving.

Marie Mardal, Petur W Dalsgaard, Brian S Rasmussen, Kristian Linnet, Christian B Mollerup.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is widely used to detect chemicals with a broad range of physiochemical properties in complex biological samples. However, the current data analysis strategies are not sufficiently scalable because of data complexity and amplitude. In this article, we report a novel data analysis strategy for HRMS data founded on structured query language database archiving. A database called ScreenDB was populated with parsed untargeted LC-HRMS data after peak deconvolution from forensic drug screening data. The data were acquired using the same analytical method over 8 years. ScreenDB currently holds data from around 40,000 data files, including forensic cases and quality control samples that can be readily sliced and diced across data layers. Long-term monitoring of system performance, retrospective data analysis for new targets, and identification of alternative analytical targets for poorly ionized analytes are examples of ScreenDB applications. These examples demonstrate that ScreenDB makes a significant improvement to forensic services and that the concept has potential for broad applications for all large-scale biomonitoring projects that rely on untargeted LC-HRMS data.

DOI: https://doi.org/10.1021/acs.analchem.2c03769
J Pharm Biomed Anal. 2023 Feb 15. pii: S0731-7085(23)00062-6. [Epub ahead of print]227 115293

Development, validation and application of a selective and sensitive LC-MS/MS method for the quantification of daptomycin in a suspension of Mammaliicoccus sciuri in Mueller-Hinton broth.

Lukas Kirchner, Tessa Marciniak, Wilma Ziebuhr, Oliver Scherf-Clavel, Ulrike Holzgrabe.

  Reports of therapy failures related to the use of daptomycin (DAP) are steadily increasing. This is mainly due to emerging DAP resistance for which, however, the underlying mechanism is often unknown. To elucidate the mode of action of novel DAP resistance traits it is indispensable to reliably detect and quantify DAP in complex matrices such as bacterial culture media. In this study, we established a selective and sensitive quantification method for DAP upon growth of a DAP resistant Mammaliicoccus sciuri strain in Mueller-Hinton medium. The method combined methanol-induced protein precipitation followed by high performance liquid chromatography with gradient elution coupled to mass spectrometry (LC-MS/MS) using daptomycin-d5 as internal standard. All validation parameters met the acceptance criteria of the European Medicines Agency (EMA) guideline on bioanalytical method validation. We successfully applied this highly selective and sensitive LC-MS/MS method for the quantification of DAP in in vitro studies addressing DAP resistance mechanisms in Gram-positive bacteria.

Keywords:  Daptomycin; High performance liquid chromatography; Mammaliicoccus sciuri; Mass spectrometry; Method validation; Mueller-Hinton medium

DOI:  https://doi.org/10.1016/j.jpba.2023.115293
Antibiotics (Basel). 2023 Jan 18. pii: 199. [Epub ahead of print]12(2):

A Validated UHPLC-MS/MS Method to Quantify Eight Antibiotics in Quantitative Dried Blood Spots in Support of Pharmacokinetic Studies in Neonates.

Qian Liu, Lanyu Liu, Yu Yuan, Feifan Xie.

   OBJECTIVES: Conduction of pharmacokinetic (PK) study in pediatric patients is challenging due to blood sampling limits. The dried blood spots (DBS) method represents a potential matrix for microsampling in support of PK studies in children. Herein, we used the Capitainer® qDBS device to develop a DBS method that can collect an exact 10 µL volume of blood on a paper card. This DBS method was developed to simultaneously quantify the concentrations of eight antibiotics, including sulbactam, tazobactam, ampicillin, meropenem, cefotaxime, cefoperazone, piperacillin, and metronidazole using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
METHODS: The prepared DBS samples were extracted in methanol containing acetaminophen as the internal standard at 20 °C on a block bath shaker at 500 rpm for 30 min. The extracted antibiotics were eluted on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) using gradient elution with a total chromatographic run time of 6.5 min. The precursor and product ions of the analytes were detected by use of the multiple reaction monitoring (MRM) mode.
RESULTS: No interfering peaks at the respective retention times of the analytes were observed in DBS samples. The lower limits of quantification (LLOQ) for the antibiotics were between 0.25 and 2.0 μg/mL, and satisfactory accuracies (intra/inter-assay bias -16.7 to +13.6%) and precisions (intra/inter-assay coefficient of variations 1.5-15.6%) were obtained for the analytes. As a proof of concept, the method was applied to DBS samples obtained from neonatal patients treated with ampicillin and piperacillin/sulbactam.
CONCLUSIONS: The DBS method is simple and robust, and it can be used in children with limited blood sampling.

Keywords:  LC–MS/MS; antibiotics; dried blood spots; pediatrics; pharmacokinetics

DOI:  https://doi.org/10.3390/antibiotics12020199
Anal Chem. 2023 Feb 23.

Analysis of a Broad Range of Carbonyl Metabolites in Exhaled Breath by UHPLC-MS.

Zhenzhen Xie, James D Morris, Stephanie J Mattingly, Saurin R Sutaria, Jiapeng Huang, Michael H Nantz, Xiao-An Fu.

Analysis of volatile organic compounds (VOCs) in exhaled breath (EB) has shown great potential for disease detection including lung cancer, infectious respiratory diseases, and chronic obstructive pulmonary disease. Although many breath sample collection and analytical methods have been developed for breath analysis, analysis of metabolic VOCs in exhaled breath is still a challenge for clinical application. Many carbonyl compounds in exhaled breath are related to the metabolic processes of diseases. This work reports a method of ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-MS) for the analysis of a broad range of carbonyl metabolites in exhaled breath. Carbonyl compounds in the exhaled breath were captured by a fabricated silicon microreactor with a micropillar array coated with 2-(aminooxy)ethyl-N,N,N-trimethylammonium (ATM) triflate. A total of six subgroups consisting of saturated aldehydes and ketones, hydroxy-aldehydes, and hydroxy-ketones, unsaturated 2-alkenals, and 4-hydroxy-2-alkenals were identified in the exhaled breath. The combination of a silicon microreactor for the selective capture of carbonyl compounds with UHPLC-MS analysis may provide a quantitative method for the analysis of carbonyls to identify disease markers in exhaled breath.

DOI: https://doi.org/10.1021/acs.analchem.2c04604
Biomolecules. 2023 Jan 27. pii: 244. [Epub ahead of print]13(2):

Metabolic Pathway Analysis: Advantages and Pitfalls for the Functional Interpretation of Metabolomics and Lipidomics Data.

Sofia Tsouka, Mojgan Masoodi.

  Over the past decades, pathway analysis has become one of the most commonly used approaches for the functional interpretation of metabolomics data. Although the approach is widely used, it is not well standardized and the impact of different methodologies on the functional outcome is not well understood. Using four publicly available datasets, we investigated two main aspects of topological pathway analysis, namely the consideration of non-human native enzymatic reactions (e.g., from microbiota) and the interconnectivity of individual pathways. The exclusion of non-human native reactions led to detached and poorly represented reaction networks and to loss of information. The consideration of connectivity between pathways led to better emphasis of certain central metabolites in the network; however, it occasionally overemphasized the hub compounds. We proposed and examined a penalization scheme to diminish the effect of such compounds in the pathway evaluation. In order to compare and assess the results between different methodologies, we also performed over-representation analysis of the same datasets. We believe that our findings will raise awareness on both the capabilities and shortcomings of the currently used pathway analysis practices in metabolomics. Additionally, it will provide insights on various methodologies and strategies that should be considered for the analysis and interpretation of metabolomics data.

Keywords:  metabolism; metabolomics; network topology; over-representation analysis; pathway analysis

DOI:  https://doi.org/10.3390/biom13020244