bims-metlip 2024-05-26 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–05–26
thirty-one papers selected by
Sofia Costa, Matterworks

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.
Reproducible mass spectrometry data processing and compound annotation in MZmine 3.
A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4'-Nitroacetophenone.
Determination of Sulfites in Dried Fruits by Paper Spray Ionization Tandem Mass Spectrometry.
New analytical methods focusing on polar metabolite analysis in mass spectrometry and NMR-based metabolomics.
Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.
Validation and application of an online extraction and liquid chromatography tandem mass spectrometry assay for the analysis of delamanid and its DM-6705 metabolite in human breast milk.
Simultaneous quantification of 11 antiepileptic drugs using limited isotope-labeled internal standards in LC-MS/MS: An accuracy assessment.
GCMS-ID: a webserver for identifying compounds from gas chromatography mass spectrometry experiments.
LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.
Rapid evaporative ionization mass spectrometry: A survey through 15 years of applications.
A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Fosfomycin Concentrations in Human Prostatic Tissue.
How Wastewater Reflects Human Metabolism─Suspect Screening of Pharmaceutical Metabolites in Wastewater Influent.
Quantification of mangiferin in streptozotocin-induced diabetic rat plasma using UPLC-MS/MS: Pharmacokinetic assessment of Gentiana rhodantha extract after oral administration.
Development of a quantification method for arginase inhibitors by LC-MS/MS with benzoyl chloride derivatization.
Innovative Solid-Phase Extraction Strategies for Improving the Advanced Chromatographic Determination of Drugs in Challenging Biological Samples.
Quantitative analysis of 34 sex (Pro)hormones, conjugates and bioactive oxidation products thereof in human plasma by GC- and LC-MS/MS and systematic investigation of overestimations of analyte concentrations not accounted for by method validation.
Rapid determination of 111 anti-infective drugs possibly added in cosmetics using high-performance liquid chromatography-tandem mass spectrometry with scheduled multiple reaction monitoring.
Data processing for high-throughput mass spectrometry in drug discovery.
Comparative Metabolomics and Microbiome Analysis of Ethanol versus OMNImet/gene•GUT Fecal Stabilization.
NMR Precision Metabolomics: Dynamic Peak Sum Thresholding and Navigators for Highly Standardized and Reproducible Metabolite Profiling of Clinical Urine Samples.
Molecularly imprinted resin modified with ionic liquid for dispersive filter extraction and determination of perfluoroalkyl acids in eggs.
Crystallization and Solvent Evaporation Ionization Mass Spectrometry (CSEI-MS) for Rapid Detection of Drugs in Complex Matrices.
Determination of chlorothalonil in vegetables/fruits using liquid chromatography-tandem mass spectrometry with a discharge adapter interface.
Analytical methods for stable isotope labeling to elucidate rapid auxin kinetics in Arabidopsis thaliana.
Electric Field-Modulated Electrospray Ionization Mass Spectrometry for Quantity Calibration and Mass Tracking.
Metabolomics for Clinical Biomarker Discovery and Therapeutic Target Identification.
A simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method for the quantitative analysis of VX-548 in monkey plasma: Method validation and application to pharmacokinetic study.
Label-Free Direct Identification of MicroRNAs Based on a Narrow Constant-Inner-Diameter Emitter Mass Spectrometry Analysis.
Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.
Comparison of Various Extraction Approaches for Optimized Preparation of Intracellular Metabolites from Human Mesenchymal Stem Cells and Fibroblasts for NMR-Based Study.

Metabolites. 2024 May 11. pii: 280. [Epub ahead of print]14(5):

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Masoumeh Dorrani, Jifang Zhao, Nihel Bekhti, Alessia Trimigno, Sangil Min, Jongwon Ha, Ahram Han, Elizabeth O'Day, Jurre J Kamphorst.

  Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.

Keywords:  HILIC; U-13C-metabolite internal standard; biomarkers; clinical metabolomics; hydrophilic interaction liquid chromatography; triple quadrupole mass spectrometry

DOI:  https://doi.org/10.3390/metabo14050280
Nat Protoc. 2024 May 20.

Reproducible mass spectrometry data processing and compound annotation in MZmine 3.

Steffen Heuckeroth, Tito Damiani, Aleksandr Smirnov, Olena Mokshyna, Corinna Brungs, Ansgar Korf, Joshua David Smith, Paolo Stincone, Nicola Dreolin, Louis-Félix Nothias, Tuulia Hyötyläinen, Matej Orešič, Uwe Karst, Pieter C Dorrestein, Daniel Petras, Xiuxia Du, Justin J J van der Hooft, Robin Schmid, Tomáš Pluskal.

Untargeted mass spectrometry (MS) experiments produce complex, multidimensional data that are practically impossible to investigate manually. For this reason, computational pipelines are needed to extract relevant information from raw spectral data and convert it into a more comprehensible format. Depending on the sample type and/or goal of the study, a variety of MS platforms can be used for such analysis. MZmine is an open-source software for the processing of raw spectral data generated by different MS platforms. Examples include liquid chromatography-MS, gas chromatography-MS and MS-imaging. These data might typically be associated with various applications including metabolomics and lipidomics. Moreover, the third version of the software, described herein, supports the processing of ion mobility spectrometry (IMS) data. The present protocol provides three distinct procedures to perform feature detection and annotation of untargeted MS data produced by different instrumental setups: liquid chromatography-(IMS-)MS, gas chromatography-MS and (IMS-)MS imaging. For training purposes, example datasets are provided together with configuration batch files (i.e., list of processing steps and parameters) to allow new users to easily replicate the described workflows. Depending on the number of data files and available computing resources, we anticipate this to take between 2 and 24 h for new MZmine users and nonexperts. Within each procedure, we provide a detailed description for all processing parameters together with instructions/recommendations for their optimization. The main generated outputs are represented by aligned feature tables and fragmentation spectra lists that can be used by other third-party tools for further downstream analysis.

DOI: https://doi.org/10.1038/s41596-024-00996-y
Biomedicines. 2024 May 02. pii: 1003. [Epub ahead of print]12(5):

A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4'-Nitroacetophenone.

Timotej Jankech, Ivana Gerhardtova, Petra Majerova, Juraj Piestansky, Lubica Fialova, Josef Jampilek, Andrej Kovac.

  Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4'-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5-8.2% and 2.3-7.4%, accuracy was in the range of 93.3-109.7% and 94.7-110.1%, limits of detection (LODs) were in the range of 0.15-9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer's disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

Keywords:  Alzheimer’s disease; derivatization; liquid chromatography; tryptophan metabolites

DOI:  https://doi.org/10.3390/biomedicines12051003
Molecules. 2024 May 08. pii: 2192. [Epub ahead of print]29(10):

Determination of Sulfites in Dried Fruits by Paper Spray Ionization Tandem Mass Spectrometry.

Donghoon Lee, Heejin Ro, Seoyoung Hwang, Minkyu Lee, Hyebeen Kim, Jaeyoung Heo, Sangwon Cha.

  Sulfite, a widely used food additive, is subject to regulated labeling. The extraction of sulfite as the stable hydroxymethylsulfonate (HMS) form and its quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized for their good sensitivity, selectivity, and versatility across various food materials. This study aimed to develop a cost-effective and simpler method for sulfite quantitation, while maintaining the superior sensitivity and selectivity of mass spectrometry (MS). To achieve this, we introduced paper spray ionization (PSI), an ambient desorption ionization technique that could achieve the direct measurement of analytes without employing separation. We also employed a novel internal standard (IS) structurally similar to the analyte, replacing the more expensive isotopically labeled IS. Although the PSI-MS/MS method developed in this study exhibited slightly lower analytical performance compared to the conventional LC-MS/MS, it remained effective for sulfite analysis in dried fruits.

Keywords:  dried fruit; hydrophilic interaction liquid chromatography; paper spray ionization; sulfite; tandem mass spectrometry

DOI:  https://doi.org/10.3390/molecules29102192
Curr Opin Chem Biol. 2024 May 20. pii: S1367-5931(24)00042-5. [Epub ahead of print]80 102466

New analytical methods focusing on polar metabolite analysis in mass spectrometry and NMR-based metabolomics.

James McCullagh, Fay Probert.

  Following in the footsteps of genomics and proteomics, metabolomics has revolutionised the way we investigate and understand biological systems. Rapid development in the last 25 years has been driven largely by technical innovations in mass spectrometry and nuclear magnetic resonance spectroscopy. However, despite the modest size of metabolomes relative to proteomes and genomes, methodological capabilities for robust, comprehensive metabolite analysis remain a major challenge. Therefore, development of new methods and techniques remains vital for progress in the field. Here, we review developments in LC-MS, GC-MS and NMR methods in the last few years that have enhanced quantitative and comprehensive metabolome coverage, highlighting the techniques involved, their technical capabilities, relative performance, and potential impact.

Keywords:  HILIC-MS; Hyphenated methods; IC-MS; Mass spectrometry; Methods; Mixed mode; NMR; Pure-shift NMR; Quantification; Ultrafast NMR

DOI:  https://doi.org/10.1016/j.cbpa.2024.102466
Biomed Chromatogr. 2024 May 23. e5903

Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.

Marco Orleni, Robert A Parise, Julianne L Holleran, Jennifer E Amengual, Ye Feng, Timothy Synold, Jan H Beumer.

  To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 μL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4 months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

Keywords:  LC–MS/MS; human plasma; pharmacokinetics; tazemetostat; validation

DOI:  https://doi.org/10.1002/bmc.5903
J Pharm Biomed Anal. 2024 May 16. pii: S0731-7085(24)00265-6. [Epub ahead of print]246 116225

Validation and application of an online extraction and liquid chromatography tandem mass spectrometry assay for the analysis of delamanid and its DM-6705 metabolite in human breast milk.

Buyisile Mkhize, Richard Court, Sandra Castel, Anton Joubert, Marthinus van der Merwe, Gary Maartens, Francesca Conradie, Lubbe Wiesner.

  We developed and validated a bioanalytical assay to quantify delamanid and its key metabolite (DM-6705) in breast milk and aimed to quantify the secretion of these compounds in breast milk. Due to the hydrophobic nature of the analytes, special care was taken during sample preparation to prevent the formation of fatty deposits during protein precipitation. This was followed by online solid phase extraction and liquid chromatography with tandem mass spectrometry for detection. A Restek Viva BiPh C18 column (1.0 mm×50 mm, 5 µm) was used for extraction while chromatographic separation was performed using a Waters Xterra MS C18 (2.1 mm×100 mm, 5 μm) analytical column with an isocratic mobile phase consisting of acetonitrile, methanol, and 5 mM ammonium carbonate. The mass spectrometric detection of the analytes was performed using an AB Sciex 3200 mass spectrometer employing electrospray ionisation in the positive mode with multiple reaction motoring of the relevant precursor and product ions. Delamanid-d4 and OPC-14714 were used as internal standards. A quadratic (weighted 1/x concentration) regression was used to fit calibration curves for delamanid and DM-6705 over the concentration range of 10.0 - 1000 ng/mL. The intra- and inter-day validation accuracies of the quality control samples were between 92.1% and 98.3% for delamanid, and 97.0% and 102.8% for DM-6705. The percentage coefficient of variation (precision) was less than 7.8%. To our knowledge, this is the first report describing the concentrations of delamanid and DM-6705 in the breast milk of patients treated for rifampicin-resistant tuberculosis.

Keywords:  Breast milk; DM-6705; Delamanid; Drug-resistant tuberculosis; Liquid chromatography tandem mass spectrometry; Online solid phase extraction

DOI:  https://doi.org/10.1016/j.jpba.2024.116225
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 May 10. pii: S1570-0232(24)00151-X. [Epub ahead of print]1240 124143

Simultaneous quantification of 11 antiepileptic drugs using limited isotope-labeled internal standards in LC-MS/MS: An accuracy assessment.

Mariam M Abady, Ji-Seon Jeong, Ha-Jeong Kwon.

  This study aims to establish an LC-MS/MS method to simultaneously analyze 11 antiepileptic drugs with a particular focus on maintaining accuracy while reducing the number of isotope-labeled internal standards employed for cost-effectiveness. By applying a water/acetonitrile gradient elution containing 0.1 % formic acid and 2 mM ammonium formate as the mobile phase, optimal sensitivity for the target drugs could be obtained in positive ESI mode in LC-MS/MS. After optimizing various extraction techniques, extraction with 70 % acetonitrile was selected as it provided good recoveries (>93 %) for all targets without matrix effects. Accuracies within 3 % were achieved from the combination of six internal standards, while accuracies of 5 % and 10 % were obtained by reducing the number of internal standards to four and two, respectively, for more economical analysis. The accuracy of the established method was maintained in hyperglycemia, hyperlipidemia, and hyperalbuminemia sera, suggesting that it can be successfully applied to individual serum samples with various properties.

Keywords:  Accuracy assessment; Antiepileptic drugs; Internal standards; LC-MS/MS; Sample preparation procedures

DOI:  https://doi.org/10.1016/j.jchromb.2024.124143
Nucleic Acids Res. 2024 May 23. pii: gkae425. [Epub ahead of print]

GCMS-ID: a webserver for identifying compounds from gas chromatography mass spectrometry experiments.

Julia Wakoli, Afia Anjum, Tanvir Sajed, Eponine Oler, Fei Wang, Vasuk Gautam, Marcia LeVatte, David S Wishart.

GCMS-ID (Gas Chromatography Mass Spectrometry compound IDentifier) is a webserver designed to enable the identification of compounds from GC-MS experiments. GC-MS instruments produce both electron impact mass spectra (EI-MS) and retention index (RI) data for as few as one, to as many as hundreds of different compounds. Matching the measured EI-MS, RI or EI-MS + RI data to experimentally collected EI-MS and/or RI reference libraries allows facile compound identification. However, the number of available experimental RI and EI-MS reference spectra, especially for metabolomics or exposomics-related studies, is disappointingly small. Using machine learning to accurately predict the EI-MS spectra and/or RIs for millions of metabolomics and/or exposomics-relevant compounds could (partially) solve this spectral matching problem. This computational approach to compound identification is called in silico metabolomics. GCMS-ID brings this concept of in silico metabolomics closer to reality by intelligently integrating two of our previously published webservers: CFM-EI and RIpred. CFM-EI is an EI-MS spectral prediction webserver, and RIpred is a Kovats RI prediction webserver. We have found that GCMS-ID can accurately identify compounds from experimental RI, EI-MS or RI + EI-MS data through matching to its own large library of >1 million predicted RI/EI-MS values generated for metabolomics/exposomics-relevant compounds. GCMS-ID can also predict the RI or EI-MS spectrum from a user-submitted structure or annotate a user-submitted EI-MS spectrum. GCMS-ID is freely available at https://gcms-id.ca/.

DOI: https://doi.org/10.1093/nar/gkae425
Biomolecules. 2024 May 06. pii: 558. [Epub ahead of print]14(5):

LC-HRMS and GC-MS Profiling of Urine Free Cortisol, Cortisone, 6Β-, and 18-Hydroxycortisol for the Evaluation of Glucocorticoid and Mineralocorticoid Disorders.

Gregori Casals, María Antonieta Ballesteros, Angielys Zamora, Irene Martínez, Guillermo Fernández-Varo, Mireia Mora, Felicia A Hanzu, Manuel Morales-Ruiz.

   INTRODUCTION: Urine free cortisol measurements are routinely performed to evaluate hypercortisolism. Despite their analytical inaccuracy, immunoassay-based methods are frequently used. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) facilitate the incorporation of powerful diagnostic tools into clinical laboratories. In addition to its high analytical specificity and simultaneous analysis of different metabolites, accurate mass measurement allows for untargeted compound identification, which may help to identify clinically relevant metabolites or drugs.
METHODS: The present study aimed to validate a simple routine LC-HRMS method to quantify cortisol, cortisone, 6β-hydroxycortisol, and 18-hydroxycortisol simultaneously in human urine. Additionally, the study also validated a GC-MS method for the same steroids, evaluated their cross-reactivity with commercial cortisol immunoassays, and quantified the 24 h urine excretion in patients under clinical suspicion or follow-up for hypercortisolism.
RESULTS: The LC-HRMS method involved liquid-liquid extraction using dichloromethane, micro-LC for chromatographic separation and detection using the accurate masses of the steroids, and simultaneous high-resolution full scan acquisition. The method presented acceptable linearity, precision, and accuracy. Significant interference from 6β-hydroxycortisol and cortisone was demonstrated in the cortisol immunoassays, which impacted their reliability in the follow-up of patients with hypercortisolism and significant changes in these cortisol metabolites (i.e., due to drug-induced changes in CYP3A4 activity).
CONCLUSION: A rapid and accurate routine LC-HRMS method was validated, which is useful for the evaluation of hypercortisolism and other disorders of glucocorticoid and mineralocorticoid metabolism.

Keywords:  Cushing’s syndrome; cortisone; high-resolution mass spectrometry; micro-liquid chromatography; urine free cortisol

DOI:  https://doi.org/10.3390/biom14050558
J Sep Sci. 2024 May;47(9-10): e2400155

Rapid evaporative ionization mass spectrometry: A survey through 15 years of applications.

Cinzia Cafarella, Domenica Mangraviti, Francesca Rigano, Paola Dugo, Luigi Mondello.

  Rapid evaporative ionization mass spectrometry (REIMS) is a relatively recent MS technique explored in many application fields, demonstrating high versatility in the detection of a wide range of chemicals, from small molecules (phenols, amino acids, di- and tripeptides, organic acids, and sugars) to larger biomolecules, that is, phospholipids and triacylglycerols. Different sampling devices were used depending on the analyzed matrix (liquid or solid), resulting in distinct performances in terms of automation, reproducibility, and sensitivity. The absence of laborious and time-consuming sample preparation procedures and chromatographic separations was highlighted as a major advantage compared to chromatographic methods. REIMS was successfully used to achieve a comprehensive sample profiling according to a metabolomics untargeted analysis. Moreover, when a multitude of samples were available, the combination with chemometrics allowed rapid sample differentiation and the identification of discriminant features. The present review aims to provide a survey of literature reports based on the use of such analytical technology, highlighting its mode of operation in different application areas, ranging from clinical research, mostly focused on cancer diagnosis for the accurate identification of tumor margins, to the agri-food sector aiming at the safeguard of food quality and security.

Keywords:  chemometrics; direct‐MS; fingerprinting; machine learning; metabolomics

DOI:  https://doi.org/10.1002/jssc.202400155
Pharmaceutics. 2024 May 17. pii: 681. [Epub ahead of print]16(5):

A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Fosfomycin Concentrations in Human Prostatic Tissue.

Matteo Conti, Beatrice Giorgi, Rossella Barone, Milo Gatti, Pier Giorgio Cojutti, Federico Pea.

  The aim of this study was to develop and validate a fast and sensitive bioanalytical method for the accurate quantification of fosfomycin concentrations in human prostatic tissue. The sample preparation method only required milligrams of tissue sample. Each sample was mixed with two times its weight of water and homogenized. A methanol solution that was three times the volume of the internal standard (fosfomycin-13C3) was added, followed by vortex mixing and centrifugation. After its extraction from the homogenized prostatic tissue, fosfomycin was quantified by means of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) triple quadrupole system operating in negative electrospray ionization and multiple reaction monitoring detection mode. The analytical procedure was successfully validated in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect, extraction recovery, limit of quantification, and stability, according to EMA guidelines. The validation results, relative to three QC levels, were 9.9% for both the within-day and inter-day accuracy (BIAS%); 9.8% for within-day precision; and 9.9 for between-day precision. A marked matrix effect was observed in the measurements but was corrected by normalization with the internal standard. The average total recovery was high (approximatively 97% at the three control levels). The dynamic range of the method was 0.1-20 μg/g (R2 of 0.999). Negligible carry-over was observed after the injection of highly concentrated samples. F in the sample homogenate extracts was stable at 10 °C and 4 °C for at least 24 h. In the tissue sample freeze-thaw experiments, a significant decrease in F concentrations was observed after only two cycles from -80 °C to room temperature. The novel method was successfully applied to measure fosfomycin in prostatic tissue samples collected from 105 patients undergoing prostatectomy.

Keywords:  fosfomycin; liquid chromatography-tandem mass spectrometry; micro samples; prostatic tissue; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/pharmaceutics16050681
Environ Sci Technol. 2024 May 24.

How Wastewater Reflects Human Metabolism─Suspect Screening of Pharmaceutical Metabolites in Wastewater Influent.

Corina Meyer, Michael A Stravs, Juliane Hollender.

  Pharmaceuticals and their human metabolites are contaminants of emerging concern in the aquatic environment. Most monitoring studies focus on a limited set of parent compounds and even fewer metabolites. However, more than 50% of the most consumed pharmaceuticals are excreted in higher amounts as metabolites than as parents, as confirmed by a literature analysis within this study. Hence, we applied a wide-scope suspect screening approach to identify human pharmaceutical metabolites in wastewater influent from three Swiss treatment plants. Based on consumption amounts and human metabolism data, a suspect list comprising 268 parent compounds and over 1500 metabolites was compiled. Online solid phase extraction combined with liquid chromatography coupled to high-resolution tandem mass spectrometry was used to analyze the samples. Data processing, annotation, and structure elucidation were achieved with various tools, including molecular networking as well as SIRIUS/CSI:FingerID and MetFrag for MS2 spectra rationalization. We confirmed 37 metabolites with reference standards and 16 by human liver S9 incubation experiments. More than 25 metabolites were detected for the first time in influent wastewater. Semiquantification with MS2Quant showed that metabolite to parent concentration ratios were generally lower compared to literature expectations, probably due to further metabolite transformation in the sewer system or limitations in the metabolite detection. Nonetheless, metabolites pose a large fraction to the total pharmaceutical contribution in wastewater, highlighting the need for metabolite inclusion in chemical risk assessment.

Keywords:  MetFrag; SIRIUS/CSI:FingerID; drug metabolites; high resolution mass spectrometry; human pharmaceutical metabolism; molecular network; suspect screening; wastewater

DOI:  https://doi.org/10.1021/acs.est.4c00968
Pak J Pharm Sci. 2024 Mar;37(2): 291-296

Quantification of mangiferin in streptozotocin-induced diabetic rat plasma using UPLC-MS/MS: Pharmacokinetic assessment of Gentiana rhodantha extract after oral administration.

Ying Zhao, Jian- Wu Li.

Mangiferin, a key bioactive constituent in Gentiana rhodantha, has a favorable impact on reducing blood sugar. A selective and sensitive UPLC MS/MS approach was developed for determining mangiferin in diabetic rats. Employing acetonitrile protein precipitation, chromatographic separation utilized a 2.1×50 mm, 3.5μm C18 column with a mobile phase of 0.1% formic acid aqueous and 5mM ammonium acetate (A, 45%) and acetonitrile (B, 55%) at a 0.5mL min-1 flow rate. Quantification, employing the multiple reaction monitoring (MRM) mode, focused on precursor-to-product ion transitions at m/z 447.1→271.1 for baicalin m/z and 421.0→301.0 for mangiferin. Calibration curves demonstrated linearity in the 1.00~100ng/mL range, with a lower quantification limit for rat plasma set at 1.00ng/mL. Inter- and intra-day accuracies spanned -9.1% to 8.5% and mangiferin mean recovery varied from 82.3% to 86.7%. The adeptly utilized UPLC-MS/MS approach facilitated the exploration of mangiferin pharmacokinetics in diabetic rats.
J Pharm Biomed Anal. 2024 May 08. pii: S0731-7085(24)00250-4. [Epub ahead of print]246 116210

Development of a quantification method for arginase inhibitors by LC-MS/MS with benzoyl chloride derivatization.

Eric Gosselin, Petar Pop-Damkov, Aixiang Xue, Roshini Markandu, Scott Mlynarski, Ray Finlay, Alwin Schuller, Diane Ramsden, Eric T Gangl.

  Arginase is an enzyme responsible for converting arginine, a semi-essential amino acid, to ornithine and urea. Arginine depletion suppresses immunity via multiple mechanisms including inhibition of T-cell and NK cell proliferation and activity. Arginase inhibition is therefore an attractive mechanism to potentially reverse immune suppression and thus has been explored as a therapy for oncology and respiratory indications. Small molecules targeting arginase present significant bioanalytical challenges for in vitro and in vivo characterization as inhibitors of arginase are typically hydrophilic in nature. The resulting low or negative LogD characteristics are incompatible with common analytical methods such as RP-ESI-MS/MS. Accordingly, a sensitive, high-throughput bioanalytical method was developed by incorporating benzoyl chloride derivatization to increase the hydrophobic characteristics of these polar analytes. Samples were separated by reversed phase chromatography on a Waters XBridge BEH C18 3.5 μm, 30 × 3 mm column using gradient elution. The mass spec was operated in positive mode using electrospray ionization. The m/z 434.1→176.1, 439.4→181.2, 334.9→150.0 and 339.9→150.0 for AZD0011, AZD0011 IS, AZD0011-PL and AZD0011-PL IS respectively were used for quantitation. The linear calibration range of the assay was 1.00-10,000 ng/mL with QC values of 5, 50 and 500 ng/mL. The qualified method presented herein exhibits a novel, robust analytical performance and was successfully applied to evaluate the in vivo ADME properties of boronic acid-based arginase inhibitor prodrug AZD0011 and its active payload AZD0011-PL.

Keywords:  Arginase; Benzoyl chloride; Chromatography; Derivatization; LC-MS/MS; Mass spectrometry

DOI:  https://doi.org/10.1016/j.jpba.2024.116210
Molecules. 2024 May 12. pii: 2278. [Epub ahead of print]29(10):

Innovative Solid-Phase Extraction Strategies for Improving the Advanced Chromatographic Determination of Drugs in Challenging Biological Samples.

Mohammadreza Mahdavijalal, Carmine Petio, Giovanni Staffilano, Roberto Mandrioli, Michele Protti.

  In the past few decades, considerable scientific strides have been made in the subject of drug analysis in human biological samples. However, the risk caused by incorrect drug plasma levels in patients still remains an important concern. This review paper attempts to investigate the advances made over the last ten years in common sample preparation techniques (SPT) for biological samples based on solid sorbents, including solid-phase extraction (SPE) and solid-phase micro-extraction (SPME), and in particular in the field of molecularly imprinted polymers (MIPs), including non-stimuli-responsive and stimuli-responsive adsorbents. This class of materials is known as 'smart adsorbents', exhibiting tailored responses to various stimuli such as magnetic fields, pH, temperature, and light. Details are provided on how these advanced SPT are changing the landscape of modern drug analysis in their coupling with liquid chromatography-mass spectrometry (LC-MS) analytical techniques, a general term that includes high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC), as well as any variation of MS, such as tandem (MS/MS), multiple-stage (MSn), and high-resolution (HRMS) mass spectrometry. Some notes are also provided on coupling with less-performing techniques, such as high-performance liquid chromatography with ultraviolet (HPLC-UV) and diode array detection (HPLC-DAD) detection. Finally, we provide a general review of the difficulties and benefits of the proposed approaches and the future prospects of this research area.

Keywords:  human biological fluids; mass spectrometry; multiple-stage mass spectrometry; smart adsorbents; solid-phase extraction; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/molecules29102278
Steroids. 2024 May 18. pii: S0039-128X(24)00079-5. [Epub ahead of print] 109441

Quantitative analysis of 34 sex (Pro)hormones, conjugates and bioactive oxidation products thereof in human plasma by GC- and LC-MS/MS and systematic investigation of overestimations of analyte concentrations not accounted for by method validation.

Miriam Raps, Carolin Kleider, Leane Lehmann.

  When investigating endocrine disorders, it is essential to assess a comprehensive quantitative profile of sex (pro)hormones in plasma including conjugates. Thus, the present study aimed to develop and validate a comprehensive mass spectrometry-based multimethod combining the direct analysis of unconjugated sex (pro)hormones and oxidation products thereof (by GC), as well as their sulfates and glucuronides present in higher concentrations (by LC) with the indirect quantification of glucuronides present in lower concentrations after selective glucuronide hydrolysis (by GC) and its application to plasma derived from ten pre- and postmenopausal women and men each. Even guideline-compliant validation experiments cannot completely reflect overestimation of analyte concentrations due to effects depending on the individual ratio of analytes (i.e. chemical formation of analytes or incomplete removal of interfering analytes). Thus, the extent of processes not accounted for by the calibration strategy were investigated and maximum over- or underestimations of analyte concentrations were assessed for each plasma sample individually. 34 analytes were successfully calibrated, validated (median accuracy 101.1 %, median inter-day precision 8.1 %) and 31 were detected above the detection limit in plasma samples. The sporadic maximum individual over- or underestimation of analyte concentrations amounted to less than 20 %.

Keywords:  Androgens; Conjugates; Estrogens; Human plasma; Mass spectrometry; Pregnenolone and progesterone

DOI:  https://doi.org/10.1016/j.steroids.2024.109441
Rapid Commun Mass Spectrom. 2024 Aug 15. 38(15): e9778

Rapid determination of 111 anti-infective drugs possibly added in cosmetics using high-performance liquid chromatography-tandem mass spectrometry with scheduled multiple reaction monitoring.

Longhai Jian, Jing Han, Hongliang Wen, Yiting Shen, Kai Zhang, Ling Yu, Rong Zheng, Xingsheng Peng, Liuqing Zhao, Chunye Sun.

RATIONALE: Illegal addition of anti-infective drugs to cosmetics at low concentrations has been found. The illicit addition of anti-infective drugs encompasses a wide variety of medications. The current sample purification methods are inadequate to detect all these compounds. A sensitive, wide-coverage, and weak-matrix-effect measurement method needs to be established to address this issue.
METHODS: Samples were extracted using acetonitrile, diluted 25 times, and then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect 111 anti-infective drugs. The method was validated and assessed for matrix effect before being applied to cosmetic products.
RESULTS: The calibration curves for the analytes exhibited a strong correlation coefficient (r > 0.995). The limit of detection ranged from 0.006 to 0.6 mg/kg. Matrix effects were significantly improved after a 25-fold dilution. The method was successfully applied to various cosmetics. Two of 82 samples tested contained lincomycin and miconazole, respectively.
CONCLUSIONS: The developed method is quick and reliable to analyze anti-infective drugs in cosmetics, with potential for both qualitative and quantitative analyses. It is a valuable tool for cosmetic research and development, contributing to safer and more effective cosmetic products.

DOI: https://doi.org/10.1002/rcm.9778
Expert Opin Drug Discov. 2024 May 24. 1-11

Data processing for high-throughput mass spectrometry in drug discovery.

Chang Liu, Hui Zhang.

   INTRODUCTION: High-throughput mass spectrometry that could deliver > 10 times faster sample readout speed than traditional LC-based platforms has emerged as a powerful analytical technique, enabling the rapid analysis of complex biological samples. This increased speed of MS data acquisition has brought a critical demand for automatic data processing capabilities that should match or surpass the speed of data acquisition. Those data processing capabilities should serve the different requirements of drug discovery workflows.
AREAS COVERED: This paper introduced the key steps of the automatic data processing workflows for high-throughput MS technologies. Specific examples and requirements are detailed for different drug discovery applications.
EXPERT OPINION: The demand for automatic data processing in high-throughput mass spectrometry is driven by the need to keep pace with the accelerated speed of data acquisition. The seamless integration of processing capabilities with LIMS, efficient data review mechanisms, and the exploration of future features such as real-time feedback, automatic method optimization, and AI model training is crucial for advancing the drug discovery field. As technology continues to evolve, the synergy between high-throughput mass spectrometry and intelligent data processing will undoubtedly play a pivotal role in shaping the future of high-throughput drug discovery applications.

Keywords:  High-throughput analysis; data processing; data review and visualization; drug discovery; mass spectrometry

DOI:  https://doi.org/10.1080/17460441.2024.2354871
Anal Chem. 2024 May 23.

Comparative Metabolomics and Microbiome Analysis of Ethanol versus OMNImet/gene•GUT Fecal Stabilization.

Heidi Isokääntä, Lucas Pinto da Silva, Naama Karu, Teemu Kallonen, Anna-Katariina Aatsinki, Thomas Hankemeier, Leyla Schimmel, Edgar Diaz, Tuulia Hyötyläinen, Pieter C Dorrestein, Rob Knight, Matej Orešič, Rima Kaddurah-Daouk, Alex M Dickens, Santosh Lamichhane.

Metabolites from feces provide important insights into the functionality of the gut microbiome. As immediate freezing is not always feasible in gut microbiome studies, there is a need for sampling protocols that provide the stability of the fecal metabolome and microbiome at room temperature (RT). Here, we investigated the stability of various metabolites and the microbiome (16S rRNA) in feces collected in 95% ethanol (EtOH) and commercially available sample collection kits with specific preservatives OMNImet•GUT/OMNIgene•GUT. To simulate field-collection scenarios, the samples were stored at different temperatures at varying durations (24 h + 4 °C, 24 h RT, 36 h RT, 48 h RT, and 7 days RT) and compared to aliquots immediately frozen at -80 °C. We applied several targeted and untargeted metabolomics platforms to measure lipids, polar metabolites, endocannabinoids, short-chain fatty acids (SCFAs), and bile acids (BAs). We found that SCFAs in the nonstabilized samples increased over time, while a stable profile was recorded in sample aliquots stored in 95% EtOH and OMNImet•GUT. When comparing the metabolite levels between aliquots stored at room temperature and at +4 °C, we detected several changes in microbial metabolites, including multiple BAs and SCFAs. Taken together, we found that storing samples at RT and stabilizing them in 95% EtOH yielded metabolomic results comparable to those from flash freezing. We also found that the overall composition of the microbiome did not vary significantly between different storage types. However, notable differences were observed in the α diversity. Altogether, the stability of the metabolome and microbiome in 95% EtOH provided results similar to those of the validated commercial collection kits OMNImet•GUT and OMNIgene•GUT, respectively.

DOI: https://doi.org/10.1021/acs.analchem.3c04436
Metabolites. 2024 May 10. pii: 275. [Epub ahead of print]14(5):

NMR Precision Metabolomics: Dynamic Peak Sum Thresholding and Navigators for Highly Standardized and Reproducible Metabolite Profiling of Clinical Urine Samples.

Alessia Trimigno, Nicole R Holderman, Chen Dong, Kari D Boardman, Jifang Zhao, Elizabeth M O'Day.

  Metabolomics, especially urine-based studies, offers incredible promise for the discovery and development of clinically impactful biomarkers. However, due to the unique challenges of urine, a highly precise and reproducible workflow for NMR-based urine metabolomics is lacking. Using 1D and 2D non-uniform sampled (NUS) 1H-13C NMR spectroscopy, we systematically explored how changes in hydration or specific gravity (SG) and pH can impact biomarker discovery. Further, we examined additional sources of error in metabolomics studies and identified Navigator molecules that could monitor for those biases. Adjustment of SG to 1.002-1.02 coupled with a dynamic sum-based peak thresholding eliminates false positives associated with urine hydration and reduces variation in chemical shift. We identified Navigator molecules that can effectively monitor for inconsistencies in sample processing, SG, protein contamination, and pH. The workflow described provides quality assurance and quality control tools to generate high-quality urine metabolomics data, which is the first step in biomarker discovery.

Keywords:  NMR; metabolomics; urinary biomarkers

DOI:  https://doi.org/10.3390/metabo14050275
Food Chem. 2024 May 14. pii: S0308-8146(24)01327-X. [Epub ahead of print]453 139677

Molecularly imprinted resin modified with ionic liquid for dispersive filter extraction and determination of perfluoroalkyl acids in eggs.

Yanke Lu, Hongyuan Yan, Pengfei Li, Yehong Han, Shigang Shen.

  Perfluoroalkyl acids (PFAAs) are emerging pollutants that endangers food safety. Developing methods for the selective determination of trace PFAAs in complex samples remains challenging. Herein, an ionic liquid modified porous imprinted phenolic resin-dispersive filter extraction-liquid chromatography-tandem mass spectrometry (IL-PIPR-DFE-LC-MS/MS) method was developed for the determination of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in eggs. The new IL-PIPR adsorbent was prepared at room temperature, which avoids the disorder and instability of the template at high temperatures. The imprinting factor of IL-PIPR for PFOA and PFOS exceeded 7.3. DFE, combined with IL-PIPR (15 mg), was used to extract PFOA and PFOS from eggs within 15 min. The established method exhibits low limits of detection (0.01-0.02 ng/g) and high recoveries (84.7%-104.7%), which surpass those of previously reported methods. This work offers a new approach to explore advanced imprinted adsorbents for PFAAs, efficient sample pretreatment technique, and analytical method for pollutants in foods.

Keywords:  Dispersive filter extraction; Egg; Liquid chromatography-tandem mass spectrometry; Molecularly imprinted resin; Perfluoroalkyl acids

DOI:  https://doi.org/10.1016/j.foodchem.2024.139677
Anal Chem. 2024 May 21.

Crystallization and Solvent Evaporation Ionization Mass Spectrometry (CSEI-MS) for Rapid Detection of Drugs in Complex Matrices.

Weiwei Chen, Qing Li, Jing Luo, Yuanjiang Pan, Hongru Feng.

Illegal addition of drugs is common but seriously threatens public health safety. Conventional mass spectrometry methods are difficult to realize direct analysis of drugs existing in some complex matrices such as seawater or soil due to the ion suppression effect and contamination to MS parts caused by nonvolatile salts. In this work, a novel crystallization and solvent evaporation ionization mass spectrometry (CSEI-MS) method was constructed and developed to achieve rapid desalting detection. CSEI only consists of a heated plate and a nebulizer and exhibits excellent desalting performance, enabling direct analysis of six drugs dissolved in eight kinds of salt solutions (up to 200 mmol/L) and three complex salty matrices. Under optimized conditions, CSEI-MS presents high sensitivity, accuracy, linearity, and intraday and interday precision. Finally, this method is applied to the quantitative analysis of drugs in seawater, hand cream, and soil. Furthermore, the highly sensitive detection of CSEI-MS is demonstrated to remain even if the detection processes are conducted within 5 s via common commercial tools.

DOI: https://doi.org/10.1021/acs.analchem.4c01469
Rapid Commun Mass Spectrom. 2024 Aug 15. 38(15): e9770

Determination of chlorothalonil in vegetables/fruits using liquid chromatography-tandem mass spectrometry with a discharge adapter interface.

Wing-Ming Chou, Wen-Ling Lin, Jenn-Feng Sheen.

RATIONALE: Chlorothalonil (CHT), a broad-spectrum fungicide, has been employed widely to control foliar diseases, whereas with a major metabolite of polar 4-hydroxychlorothalonil (CHT-4-OH), only an acceptable nonpolar CHT residue is allowed by most countries. This study involves the method development for CHT residue in vegetables/fruits using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a novel modified discharge-adaptor (DA) interface.
METHODS: CHT residue was analyzed using LC-MS/MS with DA interface (LC-DA-MS/MS), developed in our previous works. A DA was placed on the electrospray tip to switch the ionization modes. A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was applied to extract CHT residue of vegetables/fruits efficiently with less sample preparation time and analysis cost.
RESULTS: CHT and CHT-4-OH spiked in four different vegetables/fruits were extracted using the modified QuEChERS method. After LC with isocratic elution, CHT and CHT-4-OH were separated within 3 min. Using LC-DA-MS/MS, the ion signals of CHT were improved two to three times, and the limit of quantification of 5 ng/g and linearity (r2 > 0.99) in the range of 5-200 ng/g were achieved using 10 g of vegetables/fruits. The precision and accuracy were within 15% each. The modified QuEChERS and LC-DA-MS/MS were applied to examine eight field-grown vegetables/fruits; 9.5 and 2588.9 ng/g of CHT were detected in two vegetables/fruits.
CONCLUSION: LC-DA-MS/MS combined with modified QuEChERS was successfully applied to determine CHT residue <10 ng/g in vegetables/fruits and with satisfied validation results. The developed method could reduce both analysis cost and time, attributing to simplifications in modified QuEChERS, isocratic elution, and DA interface in LC-DA-MS/MS.

DOI: https://doi.org/10.1002/rcm.9770
PLoS One. 2024 ;19(5): e0303992

Analytical methods for stable isotope labeling to elucidate rapid auxin kinetics in Arabidopsis thaliana.

Qian Tang, Molly Tillmann, Jerry D Cohen.

The phytohormone auxin plays a critical role in plant growth and development. Despite significant progress in elucidating metabolic pathways of the primary bioactive auxin, indole-3-acetic acid (IAA), over the past few decades, key components such as intermediates and enzymes have not been fully characterized, and the dynamic regulation of IAA metabolism in response to environmental signals has not been completely revealed. In this study, we established a protocol employing a highly sensitive liquid chromatography-mass spectrometry (LC-MS) instrumentation and a rapid stable isotope labeling approach. We treated Arabidopsis seedlings with two stable isotope labeled precursors ([13C6]anthranilate and [13C8, 15N1]indole) and monitored the label incorporation into proposed indolic compounds involved in IAA biosynthetic pathways. This Stable Isotope Labeled Kinetics (SILK) method allowed us to trace the turnover rates of IAA pathway precursors and product concurrently with a time scale of seconds to minutes. By measuring the entire pathways over time and using different isotopic tracer techniques, we demonstrated that these methods offer more detailed information about this complex interacting network of IAA biosynthesis, and should prove to be useful for studying auxin metabolic network in vivo in a variety of plant tissues and under different environmental conditions.

DOI: https://doi.org/10.1371/journal.pone.0303992
J Am Soc Mass Spectrom. 2024 May 24.

Electric Field-Modulated Electrospray Ionization Mass Spectrometry for Quantity Calibration and Mass Tracking.

Pin-Chieh Hsu, Pawel L Urban.

Analyses conducted by electrospray ionization (ESI) mass spectrometry (MS) typically entail performing a number of preparatory steps, which include quantity calibration and mass calibration. Quantity calibration can be affected by signal noise, while mass calibration can be affected by instrumental drift if analyses are performed over an extended period of time. Here, we present two methods for achieving these calibrations using modulation of electrospray plume by alternating electric fields and demodulating the resulting MS ion currents. For this purpose, we use an ESI source fitted with three ring electrodes between the electrospray emitter and the mass spectrometer's inlet. One of these electrodes is supplied with a sine electric signal. Optionally, a nanoESI emitter is also placed between the ring electrodes and the mass spectrometer's orifice to supply calibrant ions. The ion currents, recorded with this setup, present wave-like features. In the first variant, using a triple quadrupole mass analyzer, the ion currents are subjected to data treatment by fast Fourier transform (FFT), and the resulting FFT magnitudes are correlated with analyte concentrations to produce a calibration plot. In the second variant, using a quadrupole time-of-flight mass analyzer, the mass spectra recorded at the analyte ion current maxima are mass-checked using the m/z value of the internal standard (injected via nanoESI emitter), which appears predominantly in the time intervals corresponding to the analyte ion current minima. The setup has been characterized using simulation software and optimized. Overall, the method enables the preparation of quantity calibration plots and monitoring (minor) m/z drifts during prolonged analyses.

DOI: https://doi.org/10.1021/jasms.4c00091
Molecules. 2024 May 08. pii: 2198. [Epub ahead of print]29(10):

Metabolomics for Clinical Biomarker Discovery and Therapeutic Target Identification.

Chunsheng Lin, Qianqian Tian, Sifan Guo, Dandan Xie, Ying Cai, Zhibo Wang, Hang Chu, Shi Qiu, Songqi Tang, Aihua Zhang.

  As links between genotype and phenotype, small-molecule metabolites are attractive biomarkers for disease diagnosis, prognosis, classification, drug screening and treatment, insight into understanding disease pathology and identifying potential targets. Metabolomics technology is crucial for discovering targets of small-molecule metabolites involved in disease phenotype. Mass spectrometry-based metabolomics has implemented in applications in various fields including target discovery, explanation of disease mechanisms and compound screening. It is used to analyze the physiological or pathological states of the organism by investigating the changes in endogenous small-molecule metabolites and associated metabolism from complex metabolic pathways in biological samples. The present review provides a critical update of high-throughput functional metabolomics techniques and diverse applications, and recommends the use of mass spectrometry-based metabolomics for discovering small-molecule metabolite signatures that provide valuable insights into metabolic targets. We also recommend using mass spectrometry-based metabolomics as a powerful tool for identifying and understanding metabolic patterns, metabolic targets and for efficacy evaluation of herbal medicine.

Keywords:  biomarker; mass spectrometry; metabolomics; phenotype; targets

DOI:  https://doi.org/10.3390/molecules29102198
Biomed Chromatogr. 2024 May 23. e5907

A simple and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method for the quantitative analysis of VX-548 in monkey plasma: Method validation and application to pharmacokinetic study.

Huiying Zhang, Yonggang Chen, Jing Huang, Wenfei Sun.

  VX-548 is an orally active and highly selective NaV 1.8 inhibitor that is undergoing development for the treatment of acute pain. The aim of this study was to develop a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the measurement of VX-548 in monkey plasma. VX-548 was extracted from the plasma using acetonitrile-mediated protein precipitation. The quantitative analysis was performed on a Thermo Vantage TSQ mass spectrometer with ibrutinib as an internal standard. Chromatography was performed on a Waters ACQUITY UPLC BEH C18 column with 0.1% aqueous formic acid and acetonitrile as mobile phase. The precursor-to-product ion transitions were m/z 474.2 > 165.0 and m/z 441.2 > 138.1 for VX-548 and internal standard, respectively. This developed method was successfully validated in the concentration range of 1-1000 ng/mL. The calibration curve showed excellent linearity with a correlation coefficient of >0.999. The precision expressed as relative standard deviation (RSD) was <8.4%, whereas the accuracy denoted as relative error (RE) ranged from -5.0% to 9.1%. The mean recovery was >84%. VX-548 was stable in monkey plasma after storage under certain conditions. The validated method was successfully applied to the pharmacokinetic study of VX-548 in monkey plasma after single oral (2 mg/kg) and intravenous (1 mg/kg) administrations.

Keywords:  UPLC–MS/MS; VX‐548; bioavailability; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.5907
Anal Chem. 2024 May 22.

Label-Free Direct Identification of MicroRNAs Based on a Narrow Constant-Inner-Diameter Emitter Mass Spectrometry Analysis.

Wenmei Zhang, Guizhen Zhu, Ning Li, Liangxia Wang, Mengying Wang, Yuanyuan Wu, Yaoyao Zhao, Qin Hu, Guangsheng Guo, Xiayan Wang.

MicroRNAs (miRNAs) are a class of endogenous noncoding small RNAs that play important roles in various biological processes and diseases. Direct determination of miRNAs is a cost-efficient and accurate method for analysis. Herein, we established a novel method for the analysis of miRNAs based on a narrow constant-inner-diameter mass spectrometry emitter. We utilized the gravity-assisted sleeving etching method to prepare a constant-inner-diameter mass spectrometry emitter with a capillary inner diameter of 5.5 μm, coupled it with a high-voltage power supply and a high-resolution mass spectrometer, and used it for miRNA direct detection. The method showed high sensitivity and reproducibility for the analysis of four miRNAs, with a limit of detection of 100 nmol/L (170 amol) for the Hsa-miR-1290 analysis. Compared with commercial ion sources, our method achieved higher sensitivity for miRNA detection. In addition, we analyzed the total miRNAs in the A549 cells. The result indicated that both spiked and endogenous miRNAs could be quantified with high accuracy. As a result, this method offers a promising platform for highly sensitive and accurate miRNA analysis. Furthermore, this approach can be extended to the analysis of other small oligonucleotides and holds the potential for studying clinical samples and facilitating disease diagnosis.

DOI: https://doi.org/10.1021/acs.analchem.3c05437
Metabolites. 2024 Apr 25. pii: 246. [Epub ahead of print]14(5):

Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes.

Nils W F Meijer, Susan Zwakenberg, Johan Gerrits, Denise Westland, Arif I Ardisasmita, Sabine A Fuchs, Nanda M Verhoeven-Duif, Judith J M Jans, Fried J T Zwartkruis.

  Direct infusion-high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.

Keywords:  TCA cycle; direct infusion–high-resolution mass spectrometry; glutaminolysis; glycolysis; isotope tracing; organoids; patient material

DOI:  https://doi.org/10.3390/metabo14050246
Metabolites. 2024 May 07. pii: 268. [Epub ahead of print]14(5):

Comparison of Various Extraction Approaches for Optimized Preparation of Intracellular Metabolites from Human Mesenchymal Stem Cells and Fibroblasts for NMR-Based Study.

Slavomíra Nováková, Eva Baranovičová, Zuzana Hatoková, Gábor Beke, Janka Pálešová, Romana Záhumenská, Bibiána Baďurová, Mária Janíčková, Ján Strnádel, Erika Halašová, Henrieta Škovierová.

  Metabolomics has proven to be a sensitive tool for monitoring biochemical processes in cell culture. It enables multi-analysis, clarifying the correlation between numerous metabolic pathways. Together with other analysis, it thus provides a global view of a cell's physiological state. A comprehensive analysis of molecular changes is also required in the case of mesenchymal stem cells (MSCs), which currently represent an essential portion of cells used in regenerative medicine. Reproducibility and correct measurement are closely connected to careful metabolite extraction, and sample preparation is always a critical point. Our study aimed to compare the efficiencies of four harvesting and six extraction methods. Several organic reagents (methanol, ethanol, acetonitrile, methanol-chloroform, MTBE) and harvesting approaches (trypsinization vs. scraping) were tested. We used untargeted nuclear magnetic resonance spectroscopy (NMR) to determine the most efficient method for the extraction of metabolites from human adherent cells, specifically human dermal fibroblasts adult (HDFa) and dental pulp stem cells (DPSCs). A comprehensive dataset of 29 identified and quantified metabolites were determined to possess statistically significant differences in the abundances of several metabolites when the cells were detached mechanically to organic solvent compared to when applying enzymes mainly in the classes of amino acids and peptides for both types of cells. Direct scraping to organic solvent is a method that yields higher abundances of determined metabolites. Extraction with the use of different polar reagents, 50% and 80% methanol, or acetonitrile, mostly showed the same quality. For both HDFa and DPSC cells, the MTBE method, methanol-chloroform, and 80% ethanol extractions showed higher extraction efficiency for the most identified and quantified metabolites Thus, preparation procedures provided a cell sample processing protocol that focuses on maximizing extraction yield. Our approach may be useful for large-scale comparative metabolomic studies of human mesenchymal stem cell samples.

Keywords:  NMR; intracellular metabolites; mesenchymal stem cells

DOI:  https://doi.org/10.3390/metabo14050268