bims-metlip 2024-01-28 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–01–28
24 papers selected by
Sofia Costa, Matterworks

A Method for High Throughput Free Fatty Acids Determination in a Small Section of Bovine Liver Tissue Using Supercritical Fluid Extraction Combined with Supercritical Fluid Chromatography-Medium Vacuum Chemical Ionization Mass Spectrometry.
The Development of an Extraction Method for Simultaneously Analyzing Fatty Acids in Macroalgae Using SPE with Derivatization for LC-MS/MS.
Weighting Low-Intensity MS/MS Ions and m/z Frequency for Spectral Library Annotation.
Optimized LC-MS/MS method for quantifying insulin degludec and liraglutide in rat plasma and Tissues: Application in pharmacokinetics and biodistribution.
Simultaneous determination of multiple urine biomarkers for kidney injury using SPE combined with LC-MS/MS.
Advances in Mass Spectrometry-Based Blood Metabolomics Profiling for Non-Cancer Diseases: A Comprehensive Review.
Wide metabolite coverage LC-MS/MS assay for the diagnosis of inherited metabolic disorders in urine.
Assessing the performance of a targeted absolute quantification isotope dilution liquid chromatograhy tandem mass spectrometry assay versus a commercial nontargeted relative quantification assay for detection of three major perfluoroalkyls in human blood.
Sample preparation for suspect screening of persistent, mobile and toxic substances and their phase II metabolites in human urine by mixed-mode liquid chromatography.
Simultaneous Analysis of Organic Acids, Glycerol and Phenolic Acids in Wines Using Gas Chromatography-Mass Spectrometry.
MSdeCIpher: A Tool to Link Data from Complementary Ionization Techniques in High-Resolution GC-MS to Identify Molecular Ions.
SAND: Automated Time-Domain Modeling of NMR Spectra Applied to Metabolite Quantification.
One-phase extraction coupled with photochemical reaction allows the in-depth lipid characterization of hempseeds by untargeted lipidomics.
Detection, Quantification, and Isomer Differentiation of Per- and Polyfluoroalkyl Substances (PFAS) Using MALDI-TOF with Trapped Ion Mobility.
Strategy for Comprehensive Detection and Annotation of Gut Microbiota-Related Metabolites Based on Liquid Chromatography-High-Resolution Mass Spectrometry.
A high-throughput small volume matrix based calibration using isotope dilution liquid chromatography tandem mass spectrometry analysis for 42 per and polyfluoroalkyl substances in groundwater.
Optimizing MS-Based Multi-Omics: Comparative Analysis of Protein, Metabolite, and Lipid Extraction Techniques.
An LC-MS/MS method for serum cystatin C quantification and its comparison with two commercial immunoassays.
Bioinspired Fluorine Labeling for 19F NMR-Based Plasma Amine Profiling.
Application of the Bland-Altman and Receiver Operating Characteristic (ROC) Approaches to Study Isotope Effects in Gas Chromatography-Mass Spectrometry Analysis of Human Plasma, Serum and Urine Samples.
Establishment and optimization of a high-throughput UPLC-MS/MS method for the simultaneous quantitation of cycloicaritin and its valine carbamate prodrug in rat plasma.
Intramolecular Ring-Chain Equilibrium Elimination Strategy for Pinpointing C═C Positional and Geometric Isomers of N-Alkylpyridinium Unsaturated Fatty Acid Derivatives via Ion Mobility-Mass Spectrometry.
In Silico-Predicted Dynamic Oxlipidomics MS/MS Library: High-Throughput Discovery and Characterization of Unknown Oxidized Lipids.
Automated 2D peak detection in gas chromatography-ion mobility spectrometry through persistent homology.

Mass Spectrom (Tokyo). 2024 ;13(1): A0141

A Method for High Throughput Free Fatty Acids Determination in a Small Section of Bovine Liver Tissue Using Supercritical Fluid Extraction Combined with Supercritical Fluid Chromatography-Medium Vacuum Chemical Ionization Mass Spectrometry.

Toshinobu Hondo, Yumi Miyake, Michisato Toyoda.

  A novel ionization technique named medium vacuum chemical ionization (MVCI) mass spectrometry (MS), which is a chemical ionization using oxonium (H3O+) and hydroxide (OH-) formed from water, has excellent compatibility with the supercritical fluid extraction (SFE)/supercritical fluid chromatography (SFC). We have studied a method to determine free fatty acids (FFAs) in a small section of bovine liver tissue using SFE/SFC-MVCI MS analysis without further sample preparation. A series of FFA molecules interact with the C18 stationary phase, exhibiting broad chromatographic peaks when using a non-modified CO2 as the mobile phase. It can be optimized by adding a small content of methanol to the mobile phase as a modifier; however, it may dampen the ionization efficiency of MVCI since the proton affinity of methanol is slightly higher than water. We have carefully evaluated the modifier content on the ion detection and column efficiencies. The obtained result showed that an optimized performance was in the range of 1 to 2% methanol-modified CO2 mobile phase for both column efficiency and peak intensity. Higher methanol content than 2% degrades both peak intensity and column efficiency. Using optimized SFC conditions, a section of bovine liver tissue sliced for 14 µm thickness by 1 mm square, which is roughly estimated as about 3300 hepatocytes, was applied to determine 18 FFAs amounts for carbon chains of C12-C24. An amount of each tested FFA was estimated as in the range of 0.07 to 2.6 fmol per cell.

Keywords:  bovine liver; free fatty acids; supercritical fluid chromatography; supercritical fluid extraction; time-of-flight mass spectrometer

DOI:  https://doi.org/10.5702/massspectrometry.A0141
Molecules. 2024 Jan 16. pii: 430. [Epub ahead of print]29(2):

The Development of an Extraction Method for Simultaneously Analyzing Fatty Acids in Macroalgae Using SPE with Derivatization for LC-MS/MS.

Taewoo Yum, Eun-Yong Kim, Yeongeun Kim, Sukyoung Choi, Ki-Jung Paeng.

  Fatty acid analysis is an essential step in evaluating the potential of macroalgae for biodiesel production. An extraction method was developed to simultaneously analyze up to five types of biodiesel-fuel-related fatty acids (myristic acid, palmitic acid, cis-palmitvaccenic acid, stearic acid, and oleic acid) in macroalgae using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Lypophilization and solid-phase extraction (SPE) techniques were applied to improve the extraction efficiency and effectively purify samples. The optimal conditions for SPE were set by comparing the recoveries according to the various solvent conditions for each step (loading, washing, and elution). In addition, the introduction of trimethylaminoethyl (TMAE) derivatives to the hydroxyl group of the target analyte increased the ionization efficiency and sensitivity. The derivatized samples were analyzed using the LC-MS/MS method with electrospray ionization in the positive and multiple-reaction monitoring modes. The target analytes were separated and detected within 13.5 min using a CAPCELL PAK C18 MGII S3 column. Gradient elution was performed using distilled water and acetonitrile containing 5 mM ammonium acetate. This method offers a reliable and sensitive tool for the analysis of macroalgae samples for their potential use in biodiesel production. To the best of our knowledge, this is the first report on the simultaneous determination of fatty acids in macroalgae using LC-MS/MS with TMAE derivatization.

Keywords:  LC–ESI–MS/MS; fatty acid; macroalgae; solid-phase extraction; trimethylaminoethyl derivatives

DOI:  https://doi.org/10.3390/molecules29020430
J Am Soc Mass Spectrom. 2024 Jan 25.

Weighting Low-Intensity MS/MS Ions and m/z Frequency for Spectral Library Annotation.

Chloe Engler Hart, Tobias Kind, Pieter C Dorrestein, David Healey, Daniel Domingo-Fernández.

Calculating spectral similarity is a fundamental step in MS/MS data analysis in untargeted metabolomics experiments, as it facilitates the identification of related spectra and the annotation of compounds. To improve matching accuracy when querying an experimental mass spectrum against a spectral library, previous approaches have proposed increasing peak intensities for high m/z ranges. These high m/z values tend to be smaller in magnitude, yet they offer more crucial information for identifying the chemical structure. Here, we evaluate the impact of using these weights for identifying structurally related compounds and mass spectral library searches. Additionally, we propose a weighting approach that (i) takes into account the frequency of the m/z values within a spectral library in order to assign higher importance to the most common peaks and (ii) increases the intensity of lower peaks, similar to previous approaches. To demonstrate our approach, we applied weighting preprocessing to modified cosine, entropy, and fidelity distance metrics and benchmarked it against previously reported weights. Our results demonstrate how weighting-based preprocessing can assist in annotating the structure of unknown spectra as well as identifying structurally similar compounds. Finally, we examined scenarios in which the utilization of weights resulted in diminished performance, pinpointing spectral features where the application of weights might be detrimental.

DOI: https://doi.org/10.1021/jasms.3c00353
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jan 20. pii: S1570-0232(24)00023-0. [Epub ahead of print]1234 124015

Optimized LC-MS/MS method for quantifying insulin degludec and liraglutide in rat plasma and Tissues: Application in pharmacokinetics and biodistribution.

Jeferson Ziebarth, Camila Diedrich, Christiane Schineider Machado, Rubiana Mara Mainardes.

  We present an ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of insulin degludec (I-Deg) and liraglutide (LIRA) in rat plasma and tissues, characterized by its sensitivity and selectivity. Chromatographic separation was achieved using an Acquity UPLC BEH C18 column, leveraging a mobile phase of acetonitrile and water (both with 0.1 % formic acid) under gradient elution over a run time of 7.5 min. The mass spectrometer operated in positive electrospray ionization multiple reaction monitoring (MRM) mode, tracking transitions of m/z 1221.6 → 641.6 for I-Deg, m/z 938.7 → 1064.1 for LIRA, and m/z 1184.7 → 454.4 for the internal standard. Validation ranged from 5 to 100 ng/mL, exhibiting robust linearity (r2 > 0.99) and limits of detection (LOD) of 1.63-2.02 ng/mL for I-Deg and 0.96-1.62 ng/mL for LIRA. Limits of quantification (LOQ) were 2.38-4.76 ng/mL for I-Deg and 3.22-4.40 ng/mL for LIRA. Notably, no significant matrix effects were detected. Stability was confirmed under various conditions, and precision metrics (intraday RSD 1.68-8.05 % for I-Deg and 1.11-7.69 % for LIRA; interday RSD 1.39-8.61 % for I-Deg and 1.06-8.83 % for LIRA) alongside accuracy (90.5-114.9 % for I-Deg and 92.7-113.7 % for LIRA) were within acceptable ranges. The method was successfully applied to pharmacokinetic and biodistribution studies following simultaneous subcutaneous administration of LIRA and I-Deg in rats.

Keywords:  Bioanalytical method; Biodistribution, Pharmacokinetics; Diabetes mellitus; LC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124015
Clin Chim Acta. 2024 Jan 19. pii: S0009-8981(24)00031-7. [Epub ahead of print] 117790

Simultaneous determination of multiple urine biomarkers for kidney injury using SPE combined with LC-MS/MS.

Hewei Sun, Qing Li, Zhonggan Jin, Yide Lu, Yi Ju.

   BACKGROUND AND OBJECTIVES: Urinary biomarkers such as low molecular weight proteins and small molecular weight metabolites are crucial in the diagnosis of kidney injury. The objective of this study was to develop and preliminarily validate a sensitive and specific method using solid-phase extraction (SPE) in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous measurement of these biomarkers in human urine.
METHOD: This study presents the development of a solid-phase extraction method integrated with LC-MS/MS analyzing biomarkers including creatinine, urea, β2-microglobulin, α1-microglobulin, and cystatin C in human urine. An enhanced solid-phase cartridge technique was employed for peptide purification and dilution of small molecule metabolites during sample preparation.
RESULTS: The developed LC-MS/MS method achieved satisfactory separation of the five analytes within 15 min. Accuracy levels ranged from -8.6% to 13.6%. Both intra-assay and inter-assay imprecision rates were maintained below 7.9% for all analytes.
CONCLUSIONS: The established LC-MS/MS method effectively quantifies creatinine, urea, β2-microglobulin, α1-microglobulin and cystatin C concurrently. This offers a viable alternative for the detection of kidney injury biomarkers in human urine, demonstrating potential for clinical application in kidney injury diagnosis.

Keywords:  Kidney injury biomarkers; Liquid chromatography; Mass spectrometry; Protein absolute quantification; Solid phase extraction

DOI:  https://doi.org/10.1016/j.cca.2024.117790
Metabolites. 2024 Jan 14. pii: 54. [Epub ahead of print]14(1):

Advances in Mass Spectrometry-Based Blood Metabolomics Profiling for Non-Cancer Diseases: A Comprehensive Review.

Ekaterina Demicheva, Vladislav Dordiuk, Fernando Polanco Espino, Konstantin Ushenin, Saied Aboushanab, Vadim Shevyrin, Aleksey Buhler, Elena Mukhlynina, Olga Solovyova, Irina Danilova, Elena Kovaleva.

  Blood metabolomics profiling using mass spectrometry has emerged as a powerful approach for investigating non-cancer diseases and understanding their underlying metabolic alterations. Blood, as a readily accessible physiological fluid, contains a diverse repertoire of metabolites derived from various physiological systems. Mass spectrometry offers a universal and precise analytical platform for the comprehensive analysis of blood metabolites, encompassing proteins, lipids, peptides, glycans, and immunoglobulins. In this comprehensive review, we present an overview of the research landscape in mass spectrometry-based blood metabolomics profiling. While the field of metabolomics research is primarily focused on cancer, this review specifically highlights studies related to non-cancer diseases, aiming to bring attention to valuable research that often remains overshadowed. Employing natural language processing methods, we processed 507 articles to provide insights into the application of metabolomic studies for specific diseases and physiological systems. The review encompasses a wide range of non-cancer diseases, with emphasis on cardiovascular disease, reproductive disease, diabetes, inflammation, and immunodeficiency states. By analyzing blood samples, researchers gain valuable insights into the metabolic perturbations associated with these diseases, potentially leading to the identification of novel biomarkers and the development of personalized therapeutic approaches. Furthermore, we provide a comprehensive overview of various mass spectrometry approaches utilized in blood metabolomics research, including GC-MS, LC-MS, and others discussing their advantages and limitations. To enhance the scope, we propose including recent review articles supporting the applicability of GC×GC-MS for metabolomics-based studies. This addition will contribute to a more exhaustive understanding of the available analytical techniques. The Integration of mass spectrometry-based blood profiling into clinical practice holds promise for improving disease diagnosis, treatment monitoring, and patient outcomes. By unraveling the complex metabolic alterations associated with non-cancer diseases, researchers and healthcare professionals can pave the way for precision medicine and personalized therapeutic interventions. Continuous advancements in mass spectrometry technology and data analysis methods will further enhance the potential of blood metabolomics profiling in non-cancer diseases, facilitating its translation from the laboratory to routine clinical application.

Keywords:  GC×GC-MS; biomarkers; blood profiling; glycomics; lipidomics; mass spectrometry; metabolomics; non-cancer diseases; review

DOI:  https://doi.org/10.3390/metabo14010054
Talanta. 2024 Jan 19. pii: S0039-9140(24)00078-X. [Epub ahead of print]271 125699

Wide metabolite coverage LC-MS/MS assay for the diagnosis of inherited metabolic disorders in urine.

Eliška Ivanovová, Barbora Piskláková, Dana Dobešová, Hana Janečková, Hana Foltenová, Aleš Kvasnička, Matúš Prídavok, Kateřina Bouchalová, Julie de Sousa, David Friedecký.

   OBJECTIVE: The laboratory diagnosis of inherited metabolic disorders (IMD) has undergone significant development in recent decades, mainly due to the use of mass spectrometry, which allows rapid multicomponent analysis of a wide range of metabolites. Combined with advanced software tools, the diagnosis becomes more efficient as a benefit for both physicians and patients.
METHODS: A hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry assay for determination of urinary purines, pyrimidines, N-acylglycines, N-acetylated amino acids, sugars, sugar alcohols and other diagnostically important biomarkers was developed and validated. Evaluation of the results consisting of utilisation of robust scaling and advanced visualization tools is simple and even suitable for urgent requirements.
RESULTS: The developed method, covering 65 biomarkers, provides a comprehensive diagnostic platform for 51 IMD. For most analytes, linearity with R2 > 0.99, intra and inter-day accuracy between 80 and 120 % and precision lower than 20 % were achieved. Diagnostic workflow was evaluated on 47 patients and External Quality Assurance samples involving a total of 24 different IMD. Over seven years, more than 2300 urine samples from patients suspected for IMD have been routinely analysed.
CONCLUSIONS: This method offers the advantage of a broad coverage of intermediate metabolites of interest and therefore may be a potential alternative and simplification for clinical laboratories that use multiple methods for screening these markers.

Keywords:  Diagnosis; Hydrophilic interaction chromatography; Inherited metabolic disorders; Liquid chromatography; Mass spectrometry

DOI:  https://doi.org/10.1016/j.talanta.2024.125699
J Mass Spectrom. 2024 Feb;59(2): e4999

Assessing the performance of a targeted absolute quantification isotope dilution liquid chromatograhy tandem mass spectrometry assay versus a commercial nontargeted relative quantification assay for detection of three major perfluoroalkyls in human blood.

Samira Salihovic, Linda Dunder, Monica Lind, Lars Lind.

  Isotope dilution ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is commonly used for trace analysis of polyfluoroalkyl and perfluoroalkyl substances (PFAS) in difficult matrices. Commercial nontargeted analysis of major PFAS where relative concentrations are obtained cost effectively is rapidly emerging and is claimed to provide comparable results to that of absolute quantification using matrix matched calibration and isotope dilution UHPLC-MS/MS. However, this remains to be demonstrated on a large scale. We aimed to assess the performance of a targeted absolute quantification isotope dilution LC-MS/MS assay versus a commercial nontargeted relative quantification assay for detection of three major PFAS in human blood. We evaluated a population-based cohort of 503 individuals. Correlations were assessed using Spearman's rank correlation coefficients (rho). Precision and bias were assessed using Bland-Altman plots. For perfluorooctane sulfonic acid, the median concentrations were 5.10 ng/mL (interquartile range [IQR] 3.50-7.24 ng/mL), the two assays correlated with rho 0.83. For perfluorooctanoic acid, the median concentrations were 2.14 ng/mL (IQR 1.60-3.0 ng/mL), the two assays correlated with rho 0.92. For perfluorohexanesulfonate, the median concentrations were 5.5 ng/mL (IQR 2.50-11.61 ng/mL), the two assays correlated with rho 0.96. The Bland-Altman statistical test showed agreement of the mean difference for the majority of samples (97-98%) between the two assays. Absolute plasma concentrations of PFAS obtained using matrix matched calibration and isotope dilution UHPLC-MS/MS show agreement with relative plasma concentrations from a nontargeted commercial platform by Metabolon. We observed striking consistency between the two assays when examining the associations of the three PFAS with cholesterol, offering additional confidence in the validity of utilizing the nontargeted approach for correlations with various health phenotypes.

Keywords:  PFAS; cholesterol; isotope dilution; mass spectrometry; metabolon; nontargeted; targeted

DOI:  https://doi.org/10.1002/jms.4999
Talanta. 2024 Jan 18. pii: S0039-9140(24)00077-8. [Epub ahead of print]271 125698

Sample preparation for suspect screening of persistent, mobile and toxic substances and their phase II metabolites in human urine by mixed-mode liquid chromatography.

Mikel Musatadi, Iker Alvarez-Mora, Ines Baciero-Hernandez, Ailette Prieto, Eneritz Anakabe, Maitane Olivares, Nestor Etxebarria, Olatz Zuloaga.

  Persistent, mobile and toxic substances have drawn attention nowadays due to their particular properties, but they are overlooked in human monitorization works, limiting the knowledge of the human exposome. In that sense, human urine is an interesting matrix since not only parent compounds are eliminated, but also their phase II metabolites that could act as biomarkers. In this work, 11 sample preparation procedures involving preconcentration were tested to ensure maximum analytical coverage in human urine using mixed-mode liquid chromatography coupled with high-resolution tandem mass spectrometry. The optimized procedure consisted of a combination of solid-phase extraction and salt-assisted liquid-liquid extraction and it was employed for suspect screening. Additionally, a non-discriminatory dilute-and-shoot approach was also evaluated. After evaluating the workflow in terms of limits of identification and type II errors (i.e., false negatives), a pooled urine sample was analysed. From a list of 1450 suspects and in-silico simulated 1568 phase II metabolites (i.e. sulphates, glucuronides, and glycines), 44 and 14 substances were annotated, respectively. Most of the screened suspects were diverse industrial chemicals, but biocides, natural products and pharmaceuticals were also detected. Lastly, the complementarity of the sample preparation procedures, columns, and analysis conditions was assessed. As a result, dilute-and-shoot and the Acclaim Trinity P1 column at pH = 3 (positive ionization) and pH = 7 (negative ionization) allowed the maximum coverage since almost 70 % of the total suspects could be screened using those conditions.

Keywords:  Exposome; High-resolution tandem mass spectrometry; Mixed-mode liquid-chromatography; Mobile and toxic substances; Persistent; Phase II metabolites; Suspect screening

DOI:  https://doi.org/10.1016/j.talanta.2024.125698
Foods. 2024 Jan 05. pii: 186. [Epub ahead of print]13(2):

Simultaneous Analysis of Organic Acids, Glycerol and Phenolic Acids in Wines Using Gas Chromatography-Mass Spectrometry.

Violeta Garcia-Viñola, Candela Ruiz-de-Villa, Jordi Gombau, Montse Poblet, Albert Bordons, Cristina Reguant, Nicolas Rozès.

  Fermented beverages, particularly wines, exhibit variable concentrations of organic and phenolic acids, posing challenges in their accurate determination. Traditionally, enzymatic methods or chromatographic analyses, mainly high-performance liquid chromatography (HPLC), have been employed to quantify these compounds individually in the grape must or wine. However, chromatographic analyses face limitations due to the high sugar content in the grape must. Meanwhile, phenolic acids, found in higher quantities in red wines than in white wines, are typically analyzed using HPLC. This study presents a novel method for the quantification of organic acids (OAs), glycerol, and phenolic acids in grape musts and wines. The approach involves liquid-liquid extraction with ethyl acetate, followed by sample derivatization and analysis using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) detection mode. The results indicated successful detection and quantification of all analyzed compounds without the need for sample dilution. However, our results showed that the method of adding external standards was more suitable for quantifying wine compounds, owing to the matrix effect. Furthermore, this method is promising for quantifying other metabolites present in wines, depending on their extractability with ethyl acetate. Fermented beverages, particularly wines, exhibit variable concentrations of organic and phenolic acids, posing challenges in their accurate determination. Traditionally, enzymatic methods or chromatographic analyses, mainly high-performance liquid chromatography (HPLC), have been employed to quantify these compounds individually in the grape must or wine. The approach of this proposed method involves (i) methoximation of wine compounds in a basic medium, (ii) acidification with HCl, (iii) liquid-liquid extraction with ethyl acetate, and (iv) silyl derivatization to analyze samples with gas chromatography-mass spectrometry (GC-MS) in ion monitoring detection mode (SIM). The results indicated successful detection and quantification of all analyzed compounds without the need for sample dilution. However, our results showed that the method of adding external standards was more suitable for quantifying wine compounds, owing to the matrix effect. Furthermore, this method is promising for quantifying other metabolites present in wines, depending on their extractability with ethyl acetate. In other words, the proposed method may be suitable for profiling (targeted) or fingerprinting (untargeted) strategies to quantify wine metabolites or to classify wines according to the type of winemaking process, grape, or fermentation.

Keywords:  caffeic acid; ferulic acid; fumaric acid; gallic acid; glycerol; glyoxylic acid; oxaloacetic acid; pyruvic acid; sorbic acid; tartaric acid

DOI:  https://doi.org/10.3390/foods13020186
Metabolites. 2023 Dec 22. pii: 10. [Epub ahead of print]14(1):

MSdeCIpher: A Tool to Link Data from Complementary Ionization Techniques in High-Resolution GC-MS to Identify Molecular Ions.

Daniel Stettin, Georg Pohnert.

  Electron ionization (EI) and molecular ion-generating techniques like chemical ionization (CI) are complementary ionization methods in gas chromatography (GC)-mass spectrometry (MS). However, manual curation effort and expert knowledge are required to correctly assign molecular ions to fragment spectra. MSdeCIpher is a software tool that enables the combination of two separate datasets from fragment-rich spectra, like EI-spectra, and soft ionization spectra containing molecular ion candidates. Using high-resolution GC-MS data, it identifies and assigns molecular ions based on retention time matching, user-defined adduct/neutral loss criteria, and sum formula matching. To our knowledge, no other freely available or vendor tool is currently capable of combining fragment-rich and soft ionization datasets in this manner. The tool's performance was evaluated on three test datasets. When molecular ions are present, MSdeCIpher consistently ranks the correct molecular ion for each fragment spectrum in one of the top positions, with average ranks of 1.5, 1, and 1.2 in the three datasets, respectively. MSdeCIpher effectively reduces candidate molecular ions for each fragment spectrum and thus enables the usage of compound identification tools that require molecular masses as input. It paves the way towards rapid annotations in untargeted analysis with high-resolution GC-MS.

Keywords:  analyte identification; gas chromatography (GC); high-resolution mass spectrometry (HRMS); ionization technique; molecular ion; software tool

DOI:  https://doi.org/10.3390/metabo14010010
Anal Chem. 2024 Jan 25.

SAND: Automated Time-Domain Modeling of NMR Spectra Applied to Metabolite Quantification.

Yue Wu, Omid Sanati, Mario Uchimiya, Krish Krishnamurthy, Jonathan Wedell, Jeffrey C Hoch, Arthur S Edison, Frank Delaglio.

Developments in untargeted nuclear magnetic resonance (NMR) metabolomics enable the profiling of thousands of biological samples. The exploitation of this rich source of information requires a detailed quantification of spectral features. However, the development of a consistent and automatic workflow has been challenging because of extensive signal overlap. To address this challenge, we introduce the software Spectral Automated NMR Decomposition (SAND). SAND follows on from the previous success of time-domain modeling and automatically quantifies entire spectra without manual interaction. The SAND approach uses hybrid optimization with Markov chain Monte Carlo methods, employing subsampling in both time and frequency domains. In particular, SAND randomly divides the time-domain data into training and validation sets to help avoid overfitting. We demonstrate the accuracy of SAND, which provides a correlation of ∼0.9 with ground truth on cases including highly overlapped simulated data sets, a two-compound mixture, and a urine sample spiked with different amounts of a four-compound mixture. We further demonstrate an automated annotation using correlation networks derived from SAND decomposed peaks, and on average, 74% of peaks for each compound can be recovered in single clusters. SAND is available in NMRbox, the cloud computing environment for NMR software hosted by the Network for Advanced NMR (NAN). Since the SAND method uses time-domain subsampling (i.e., random subset of time-domain points), it has the potential to be extended to a higher dimensionality and nonuniformly sampled data.

DOI: https://doi.org/10.1021/acs.analchem.3c03078
Talanta. 2024 Jan 17. pii: S0039-9140(24)00065-1. [Epub ahead of print]271 125686

One-phase extraction coupled with photochemical reaction allows the in-depth lipid characterization of hempseeds by untargeted lipidomics.

Andrea Cerrato, Sara Elsa Aita, Giuseppe Cannazza, Chiara Cavaliere, Alberto Cavazzini, Cinzia Citti, Carmela Maria Montone, Enrico Taglioni, Aldo Laganà.

  Due to their valuable nutritional content, several hemp-derived products from hempseeds have recently been placed in the market as food and food ingredients. In particular, the lipid composition of hempseeds has raised interest for their rich content in biologically active polyunsaturated fatty acids with an optimum ratio of omega-3 and omega-6 compounds. At present, however, the overall polar lipidome composition of hempseeds remains largely unknown. In the present work, an analytical platform was developed for the extraction, untargeted HRMS-based analysis, and detailed annotation of the lipid species. First, five one- and two-phase solid-liquid extraction protocols were tested and compared on a hempseed pool sample to select the method that allowed the overall highest efficiency as well as easy coupling with lipid derivatization by photochemical [2 + 2] cycloaddition with 6-azauracil. Underivatized lipids were annotated employing a data processing workflow on Compound Discoverer software that was specifically designed for polar lipidomics, whereas inspection of the MS/MS spectra of the derivatized lipids following the aza-Paternò-Büchi reaction allowed pinpointing the regiochemistry of carbon-carbon double bonds. A total of 184 lipids were annotated, i.e., 26 fatty acids and 158 phospholipids, including minor subclasses such as N-acylphosphatidylethanolamines. Once the platform was set up, the lipid extracts from nine hempseed samples from different hemp strains were characterized, with information on the regiochemistry of free and conjugated fatty acids. The overall analytical approach helped to fill a gap in the knowledge of the nutritional composition of hempseeds.

Keywords:  Aza-paternò-büchi; Cannabis sativa; Double bond location; Fatty acids; High-resolution mass spectrometry; Phospholipids

DOI:  https://doi.org/10.1016/j.talanta.2024.125686
J Am Soc Mass Spectrom. 2024 Jan 22.

Detection, Quantification, and Isomer Differentiation of Per- and Polyfluoroalkyl Substances (PFAS) Using MALDI-TOF with Trapped Ion Mobility.

Aidan J Reynolds, Abby M Smith, Tian Autumn Qiu.

Per- and polyfluoroalkyl substances (PFAS) are a class of organic compounds that have attracted global attention for their persistence in the environment, exposure to biological organisms, and their adverse health effects. There is an urgent need to develop analytical methodologies for the characterization of PFAS in various sample matrices. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) represents a chromatography-free MS method that performs laser-based ionization and in situ analysis on samples. In this study, we present PFAS analysis by MALDI-time-of-flight (TOF) MS with trapped ion mobility spectrometry (TIMS), which provides an additional dimension of gas phase separation based on the size-to-charge ratios. MALDI matrix composition and key instrument parameters were optimized to produce different ranges of calibration curves. Parts per billion (ppb) range of calibration curves were achieved for a list of legacy and alternative perfluorosulfonic acids (PFSAs) and perfluorocarboxylic acids (PFCAs), while ion mobility spectrum filtering enabled parts per trillion (ppt) range of calibration curves for PFSAs. We also successfully demonstrated the separation of three perfluorooctanesulfonic acid (PFOS) structural isomers in the gas phase using TIMS. Our results demonstrated the new development of utilizing MALDI-TOF-MS coupled with TIMS for fast, quantitative, and sensitive analysis of PFAS, paving ways to future high-throughput and in situ analysis of PFAS such as MS imaging applications.

DOI: https://doi.org/10.1021/jasms.3c00369
Anal Chem. 2024 Jan 22.

Strategy for Comprehensive Detection and Annotation of Gut Microbiota-Related Metabolites Based on Liquid Chromatography-High-Resolution Mass Spectrometry.

Sijia Zheng, Wangshu Qin, Tiantian Chen, Runze Ouyang, Xiaolin Wang, Qi Li, Ying Zhao, Xinyu Liu, Difei Wang, Lina Zhou, Guowang Xu.

Gut microbiota, widely populating the mammalian gastrointestinal tract, plays an important role in regulating diverse pathophysiological processes by producing bioactive molecules. Extensive detection of these molecules contributes to probing microbiota function but is limited by insufficient identification of existing analytical methods. In this study, a systematic strategy was proposed to detect and annotate gut microbiota-related metabolites on a large scale. A pentafluorophenyl (PFP) column-based liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was first developed for high-coverage analysis of gut microbiota-related metabolites, which was verified to be stable and robust with a wide linearity range, high sensitivity, satisfactory recovery, and repeatability. Then, an informative database integrating 968 knowledge-based microbiota-related metabolites and 282 sample-sourced ones defined by germ-free (GF)/antibiotic-treated (ABX) models was constructed and subsequently used for targeted extraction and annotation in biological samples. Using pooled feces, plasma, and urine of mice for demonstration application, 672 microbiota-related metabolites were annotated, including 21% neglected by routine nontargeted peak detection. This strategy serves as a useful tool for the comprehensive capture of the intestinal flora metabolome, contributing to our deeper understanding of microbe-host interactions.

DOI: https://doi.org/10.1021/acs.analchem.3c05219
J Chromatogr A. 2024 Jan 07. pii: S0021-9673(24)00006-2. [Epub ahead of print]1716 464633

A high-throughput small volume matrix based calibration using isotope dilution liquid chromatography tandem mass spectrometry analysis for 42 per and polyfluoroalkyl substances in groundwater.

Matthew J Geiger, Jessica M Morrison, Douglas J Carmack, Sarah Y Lockwood-O'Brien, Michael C Stagliano, Timothy A Karrer.

  A novel method for the determination of per- and polyfluoroalkyl substances (PFAS) in groundwater is presented using a subsample, matrix-matched calibrators, 96-well plate solid phase extraction (SPE), and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Accuracy, precision, measurement of uncertainty (MOU), method detection limit (MDL), method quantitation limit (MQL), analytical measurement range, interferences/ion suppression, and analyte stability were determined as part of the in-house method validation. The method quantitates 42 PFAS compounds from nine different compound classes. Accuracy for the reference material (RM) and matrix spike (MS) ranged from 52.3 to 117.8 %, and precision for the MS and matrix spike duplicate (MSD) had a coefficient of variation (CV) from 2.0 % to 23.3 %. MDLs spanned from 0.07 to 1.97 ng L-1, with MQLs ranging from 0.20 to 5.90 ng L-1. Suppression studies determined that iron and manganese have effects on analytes that do not have paired isotopically labeled standards. The results from the in-house validation indicated that this Michigan Department of Health and Human Services laboratory developed test meets the necessary accuracy, precision, MDL, MQL and reporting limits requirement established by the laboratory's quality system essentials (QSEs) and select criteria from the Department of Defense (DoD) Quality Systems Manual for Environmental Laboratories and American Industrial Hygiene Association Laboratory Accreditation Program, LLC (AIHA LAP, LLC) accrediting International Standard Organization (ISO/IEC 17025:2017) check list.

Keywords:  Drinking water; Groundwater; High-throughput; PFAS; PFCs; Perfluoroalkyl and polyfluoroalkyl substances; Water

DOI:  https://doi.org/10.1016/j.chroma.2024.464633
Metabolites. 2024 Jan 03. pii: 34. [Epub ahead of print]14(1):

Optimizing MS-Based Multi-Omics: Comparative Analysis of Protein, Metabolite, and Lipid Extraction Techniques.

Jeong-Hun Mok, Minjoong Joo, Seonghyeon Cho, Van-An Duong, Haneul Song, Jong-Moon Park, Hookeun Lee.

  Multi-omics integrates diverse types of biological information from genomic, proteomic, and metabolomics experiments to achieve a comprehensive understanding of complex cellular mechanisms. However, this approach is also challenging due to technical issues such as limited sample quantities, the complexity of data pre-processing, and reproducibility concerns. Furthermore, existing studies have primarily focused on technical performance assessment and the presentation of modified protocols through quantitative comparisons of the identified protein counts. Nevertheless, the specific differences in these comparisons have been minimally investigated. Here, findings obtained from various omics approaches were profiled using various extraction methods (methanol extraction, the Folch method, and Matyash methods for metabolites and lipids) and two digestion methods (filter-aided sample preparation (FASP) and suspension traps (S-Trap)) for resuspended proteins. FASP was found to be more effective for the identification of membrane-related proteins, whereas S-Trap excelled in isolating nuclear-related and RNA-processing proteins. Thus, FASP may be suitable for investigating the immune response and bacterial infection pathways, whereas S-Trap may be more effective for studies focused on the mechanisms of neurodegenerative diseases. Moreover, regarding the choice of extraction method, the single-phase method identified organic compounds and compounds related to fatty acids, whereas the two-phase extraction method identified more hydrophilic compounds such as nucleotides. Lipids with strong hydrophobicity, such as ChE and TG, were identified in the two-phase extraction results. These findings highlight that significant differences among small molecules are primarily identified due to the varying polarities of extraction solvents. These results, obtained by considering variables such as human error and batch effects in the sample preparation step, offer comprehensive and detailed results not previously provided by existing studies, thereby aiding in the selection of the most suitable pre-processing approach.

Keywords:  LC-MS; metabolomics; proteomics; sample preparation

DOI:  https://doi.org/10.3390/metabo14010034
Clin Chem Lab Med. 2024 Jan 23.

An LC-MS/MS method for serum cystatin C quantification and its comparison with two commercial immunoassays.

Li Zhang, Xiaerbanu Nizhamuding, Hao Zheng, Jie Zeng, Xinyi Yuan, Zijia Ma, Weiyan Zhou, Chao Zhang, Tianjiao Zhang, Chuanbao Zhang.

   OBJECTIVES: The standardization of cystatin C (CysC) measurement has received increasing attention in recent years due to its importance in estimating glomerular filtration rate (GFR). Mass spectrometry-based assays have the potential to provide an accuracy base for CysC measurement. However, a precise, accurate and sustainable LC-MS/MS method for CysC is still lacking.
METHODS: The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated.
RESULTS: No significant matrix effect or interference was observed with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method.
CONCLUSIONS: The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.

Keywords:  LC–MS/MS; cystatin C; isotope dilution; method validation; reference method; standardization

DOI:  https://doi.org/10.1515/cclm-2023-0821
Anal Chem. 2024 Jan 20.

Bioinspired Fluorine Labeling for 19F NMR-Based Plasma Amine Profiling.

Quynh Huong Duong, Eun-Jeong Kwahk, Jumi Kim, Hahyoun Park, Heyjin Cho, Hyunwoo Kim.

Metabolite profiling serves as a powerful tool that advances our understanding of biological systems, disease mechanisms, and environmental interactions. In this study, we present an approach employing 19F-nuclear magnetic resonance (19F NMR) spectroscopy for plasma amine profiling. This method utilizes a highly efficient and reliable fluorine-labeling reagent, 3,5-difluorosalicylaldehyde, which effectively emulates pyridoxal phosphate, facilitating the formation of Schiff base compounds with primary amines. The fluorine labeling allows for distinct resolution of 19F NMR signals from amine mixtures, leading to precise identification and quantification of amine metabolites in human plasma. This advancement offers valuable tools for furthering metabolomics research.

DOI: https://doi.org/10.1021/acs.analchem.3c04485
Molecules. 2024 Jan 11. pii: 365. [Epub ahead of print]29(2):

Application of the Bland-Altman and Receiver Operating Characteristic (ROC) Approaches to Study Isotope Effects in Gas Chromatography-Mass Spectrometry Analysis of Human Plasma, Serum and Urine Samples.

Dimitrios Tsikas.

  The Bland-Altman approach is one of the most widely used mathematical approaches for method comparison and analytical agreement. This work describes, for the first time, the application of Bland-Altman to study 14N/15N and 1H/2H (D) chromatographic isotope effects of endogenous analytes of the L-arginine/nitric oxide pathway in human plasma, serum and urine samples in GC-MS. The investigated analytes included arginine, asymmetric dimethylarginine, dimethylamine, nitrite, nitrate and creatinine. There was a close correlation between the percentage difference of the retention times of the isotopologs of the Bland-Altman approach and the area under the curve (AUC) values of the receiver operating characteristic (ROC) approach (r = 0.8619, p = 0.0047). The results of the study suggest that the chromatographic isotope effects in GC-MS result from differences in the interaction strengths of H/D isotopes in the derivatives with the hydrophobic stationary phase of the GC column. D atoms attenuate the interaction of the skeleton of the molecules with the lipophilic GC stationary phase. Differences in isotope effects in plasma or serum and urine in GC-MS are suggested to be due to a kind of matrix effect, and this remains to be investigated in forthcoming studies using Bland-Altman and ROC approaches.

Keywords:  Bland–Altman; GC-MS; ROC; chromatography; isotope effects; retention time

DOI:  https://doi.org/10.3390/molecules29020365
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jan 18. pii: S1570-0232(24)00025-4. [Epub ahead of print]1234 124017

Establishment and optimization of a high-throughput UPLC-MS/MS method for the simultaneous quantitation of cycloicaritin and its valine carbamate prodrug in rat plasma.

Weiping Wang, Fengxiao Li, Shuo Gan, Jiaqi Fan, Qikun Jiang, Tianhong Zhang.

  An ultra-performance liquid chromatography-tandem mass spectrometry was developed to assay the concentration for the quantification of cycloicaritin and its carbamate prodrug (3-O-L-valyl carbamate prodrug of cycloicaritin) in the plasma of Sprague-Dawley rats. Analytes were separated on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) after liquid-liquid extraction with methyl tert-butyl ether. Acetonitrile and water containing 0.1 % formic acid were the mobile phases of the method. Using electrospray ionization in the positive ion mode, the method was performed with a total run time of 2.60 min. The response of the experiments was linear over the concentration ranges from 1 to 250 ng/mL for cycloicaritin and 1-250 ng/mL for prodrug. The intra- and inter-day precision and accuracy were within the recommended limits of the FDA. The matrix effect that we observed met the criteria. The method was successfully applied to the pharmacokinetics of cycloicaritin and its carbamate prodrug in Sprague-Dawley rats.

Keywords:  Carbamate prodrug; Cycloicaritin; Pharmacokinetics; Ultra-performance liquid chromatography-tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jchromb.2024.124017
Anal Chem. 2024 Jan 23.

Intramolecular Ring-Chain Equilibrium Elimination Strategy for Pinpointing C═C Positional and Geometric Isomers of N-Alkylpyridinium Unsaturated Fatty Acid Derivatives via Ion Mobility-Mass Spectrometry.

Yuling Li, Jiahui Bai, Kuofeng Tseng, Xiaoqiang Zhang, Li Zhang, Jing Zhang, Wenjian Sun, Yinlong Guo.

Free unsaturated fatty acids (UFA) are key intermediates of lipid metabolism and participate in many metabolic pathways with specific biological functions. Although various fragmentation-based methods for pinpointing C═C locations in UFA were developed, the current mass spectrometry methods are difficult to simultaneously differentiate geometric isomers and positional isomers in trace samples due to low ionization efficiency, low conversion, and low resolution. Herein, an intramolecular ring-chain equilibrium elimination strategy via 4-plex stable isotope labeling dual derivatization-assisted ion mobility-mass spectrometry was developed, thereby one-pot specifically labeling C═C and carboxyl groups among the trace and unstable UFA with high sensitivity, high efficiency, and good substrate generality. It achieved fast separation of both C═C positional and geometric isomers with high resolution, which benefited from eliminating the intramolecular ring-chain equilibrium by suppressing the formation of salt bridges between free carboxyl groups and pyridine cations. 4-plex stable isotope labeling reagents showed similar reactivity, enabling high-throughput quantitative analysis of omics. This method was successfully applied for accurate and rapid identification of the UFA composition in olive oil extract. These results suggest that the developed method provides new insight for rapid characterization of UFA C═C positional and geometric isomers in complex samples to explore disease biomarkers and food quality control indicators.

DOI: https://doi.org/10.1021/acs.analchem.3c04320
Anal Chem. 2024 Jan 26.

In Silico-Predicted Dynamic Oxlipidomics MS/MS Library: High-Throughput Discovery and Characterization of Unknown Oxidized Lipids.

Zheng Zhou, Xuhui Huang, Yu-Ying Zhang, Shuang Cui, Ying Wang, Meng Dong, Dayong Zhou, Beiwei Zhu, Lei Qin.

Nontargeted lipidomics using liquid chromatography-tandem mass spectrometry can detect thousands of molecules in biological samples. However, the annotation of unknown oxidized lipids is limited to the structures present in libraries, restricting the analysis and interpretation of experimental data. Here, we describe Doxlipid, a computational tool for oxidized lipid annotation that predicts a dynamic MS/MS library for every experiment. Doxlipid integrates three key simulation algorithms to predict libraries and covers 32 subclasses of oxidized lipids from the three main classes. In the evaluation, Doxlipid achieves very high prediction and characterization performance and outperforms the current oxidized lipid annotation methods. Doxlipid, combined with a molecular network, further annotates unknown chemical analogs in the same reaction or pathway. We demonstrate the broad utility of Doxlipid by analyzing oxidized lipids in ferroptosis hepatocellular carcinoma, tissue samples, and other biological samples, substantially advancing the discovery of biological pathways at the trace oxidized lipid level.

DOI: https://doi.org/10.1021/acs.analchem.3c04459
Anal Chim Acta. 2024 Feb 08. pii: S0003-2670(24)00005-9. [Epub ahead of print]1289 342204

Automated 2D peak detection in gas chromatography-ion mobility spectrometry through persistent homology.

Hadi Parastar, Joscha Christmann, Philipp Weller.

   BACKGROUND: Gas chromatography-ion mobility spectrometry (GC-IMS) is a powerful analytical technique which has gained widespread use in a variety of fields. Detecting peaks in GC-IMS data is essential for chemical identification. Topological data analysis (TDA) has the ability to record alterations in topology throughout the entire spectrum of GC-IMS data and retain this data in diagrams known as persistence diagrams. To put it differently, TDA naturally identifies characteristics such as mountains, volcanoes, and their higher-dimensional equivalents within the original data and measures their significance.
RESULTS: In the present contribution, a novel approach based on persistent homology (a flagship technique of TDA) is suggested for automatic 2D peak detection in GC-IMS. For this purpose, two different GC-IMS data examples (urine and olive oil) are used to show the performance of the proposed method. The outputs of the algorithm are GC-IMS chromatogram with detected peaks, persistence plot showing the importance (intensity) of the detected peaks and a table with retention times (RT), drift times (DT), and persistence scores of detected peaks. The RT and DT can be used for identification of the peaks and persistence scores for quantitation. Additionally, watershed segmentation is applied to the GC-IMS images to index individual peaks and segment overlapping compounds allowing for a more accurate identification and quantification of individual peaks.
SIGNIFICANCE: Inspection of the results for GC-IMS datasets showed the accurate and reliable performance of the proposed strategy based on persistent homology for automatic 2D GC-IMS peak detection for qualitative and quantitative analysis. In addition, this approach can be easily extended to other types of hyphenated chromatographic and/or spectroscopic data.

Keywords:  Chemometrics; Gas chromatography-ion mobility spectrometry; Peak detection; Persistent homology; Topological data analysis

DOI:  https://doi.org/10.1016/j.aca.2024.342204