bims-metlip 2023-06-25 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–06–25
nineteen papers selected by
Sofia Costa, Matterworks

Machine learning liquid chromatography retention time prediction model augments the dansylation strategy for metabolite analysis of urine samples.
Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome.
Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization.
Sensitive LC-MS/MS method for the temporal profiling of bile acids, fatty acids and branched-chain alpha-keto acids in maternal plasma during pregnancy and cord blood plasma at delivery.
Capillary electrophoresis-mass spectrometry as a tool for Caenorhabditis elegans metabolomics research.
A MATLAB-based app to improve LC-MS/MS data analysis for N-linked glycan peak identification.
Vacuum differential mobility spectrometry combined with column-switching liquid chromatography- mass spectrometry for the analysis of pyrrolizidine alkaloids in tea samples.
A sensitive and rapid LC-MS/MS assay for quantitation of free payload Aur0101 from antibody drug conjugate (ADC) PYX-201 in human plasma.
Simultaneous quantitation of favipiravir and its hydroxide metabolite in human plasma and hamster matrices using a UPLC-MS/MS method.
Precolumn derivatization LC-MS/MS method to simultaneous quantitation of 10 monosaccharides in rat plasma.
Desorption electrospray ionization and matrix-assisted laser desorption/ionization as imaging approaches for biological samples analysis.
A Critical Review on Chemical Speciation of Chlorine-Produced Oxidants in Seawater. Part 3: Chromatographic- and Mass Spectrometric-Based Methodologies.
Ultra-fast and accurate electron ionization mass spectrum matching for compound identification with million-scale in-silico library.
Volume-Corrected Free Energy as a New Criterion for Structural Elucidation in Chemical-Tagging-Based Metabolomics.
A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs.
Fast profiling of primary, secondary, conjugated, and sulfated bile acids in human urine and murine feces samples.
Determination of cannabinoids in 50 μL whole blood samples by online extraction using turbulent flow chromatography and LC-HRAM-Orbitrap-MS: Application on driving under the influence of drugs cases.
Systematic comparison of local approaches for isotopically nonstationary metabolic flux analysis.
Quantitative plasma profiling by 1H NMR-based metabolomics: impact of sample treatment.

J Chromatogr A. 2023 Jun 17. pii: S0021-9673(23)00393-X. [Epub ahead of print]1705 464167

Machine learning liquid chromatography retention time prediction model augments the dansylation strategy for metabolite analysis of urine samples.

Eunwoo Choi, Won Jun Yoo, Hwa-Yong Jang, Tae-Young Kim, Sung Ki Lee, Han Bin Oh.

  Herein, a standalone software equipped with a graphic user interface (GUI) is developed to predict liquid chromatography mass spectrometry (LC-MS) retention times (RTs) of dansylated metabolites. Dansylation metabolomics strategy developed by Li et al. narrows down a vast chemical space of metabolites into the metabolites containing amines and phenolic hydroxyls. Combined with differential isotope labeling, e.g., 12C-reagent labeled individual samples spiked with a 13C-reagent labeled reference or pooled sample, LC-MS analysis of the dansylated samples enables accurate relative quantification of all labeled metabolites. Herein, the LC-RTs for dansylated metabolites are predicted using an artificial neural network (ANN) machine-learning model. For the ANN modeling, 315 dansylated urine metabolites obtained from the DnsID database are used. The ANN LC-RT prediction model was reliable, with a mean absolute deviation of 0.74 min for the 30 min LC run. In the RT model, a deviation of more than 2 min was observed in only 3.2% of the total 315 metabolites, while a deviation of 1.5 min or more was observed in 11% of the metabolites. Furthermore, it was found that the LC-RT prediction was also reliable even for metabolites containing both amine and phenolic functional groups that can undergo dansylation on either one of the two functional groups, resulting in the generation of two isomeric forms. This RT-prediction model is embedded into a user-friendly GUI and can be used for identifying nontargeted dansylated metabolites with unknown RTs, along with accurate mass measurements. Furthermore, it is demonstrated that the developed software can help identify metabolites from a urine sample of an anonymous healthy pregnant woman.

Keywords:  Artificial neural network; Dansylation; Liquid chromatography–mass spectrometry; Machine learning; Retention time

DOI:  https://doi.org/10.1016/j.chroma.2023.464167
bioRxiv. 2023 Jun 05. pii: 2023.05.31.543178. [Epub ahead of print]

Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome.

Brady G Anderson, Alexander Raskind, Rylan Hissong, Michael K Dougherty, Sarah K McGill, Ajay Gulati, Casey M Theriot, Robert T Kennedy, Charles R Evans.

Compound identification is an essential task in the workflow of untargeted metabolomics since the interpretation of the data in a biological context depends on the correct assignment of chemical identities to the features it contains. Current techniques fall short of identifying all or even most observable features in untargeted metabolomics data, even after rigorous data cleaning approaches to remove degenerate features are applied. Hence, new strategies are required to annotate the metabolome more deeply and accurately. The human fecal metabolome, which is the focus of substantial biomedical interest, is a more complex, more variable, yet lesser-investigated sample matrix compared to widely studied sample types like human plasma. This manuscript describes a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public, and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach and successfully identified several rare and novel compounds, including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable but not identifiable in the original single-dimension LC-MS data. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with commercially-available instrumentation, and should apply to any dataset requiring deeper annotation of the metabolome.

DOI: https://doi.org/10.1101/2023.05.31.543178
J Pharm Biomed Anal. 2023 Jun 14. pii: S0731-7085(23)00291-1. [Epub ahead of print]234 115522

Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization.

Pascal Schorr, Caroline S Stokes, Dietrich A Volmer.

  In this study, we report a one-pot double derivatization scheme, which used acetylation after a Diels-Alder reaction using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to improve separation efficiency and provide baseline separations of the five vitamin D metabolites 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), 3β-25-hydroxyvitamin D3 (3β-25(OH)D3), 3α-25-hydroxyvitamin D3 (3α-25(OH)D3) and vitamin D3 on a C-18 stationary phase. Vitamin D metabolites are often very challenging to measure quantitatively using mass spectrometry, due to their low serum concentration levels and low ionization efficiencies. Moreover, some of these species are isomers with virtually identical mass spectral dissociation behavior. To overcome the low ionization efficiency and unspecific fragmentation behavior, derivatization using Diels-Alder reactions with Cookson-type reagents such as PTAD are common. These derivatization reactions generally result in more complicated liquid chromatography separations, because both 6R- and 6S-isomers are formed during Diels-Alder reactions. It has been shown that separations have been particularly challenging for the 3α-25(OH)D3 and 3β-25(OH)D3 epimers. Here, we optimized the PTAD derivatization and the esterification using acetic anhydride. By utilizing the esterification catalyst 4-dimethylaminopyridine, we avoided quenching and evaporation between the two derivatization steps, but were also able to perform the esterification at room temperature without heating. The optimized one-pot double derivatization LC-MS/MS assay was validated with respect to inter/intra-day precision, accuracy, recovery and linear dynamic range and applied to metabolic fingerprinting of vitamin D3 metabolites in serum samples. The metabolites 3α-25(OH)D3, 3β-25(OH)D3 and 24,25(OH)2D3, were readily quantified in all investigated samples. The method was, in principle, also fit for purpose for quantification of the native vitamin D3 species; the relatively high blank concentration of the commercial vitamin D-depleted serum used for calibration, however, limited the limits of quantification for this metabolite. The method provided insufficient limits of quantification for serum levels of 1,25(OH)2D3.

Keywords:  Derivatization; Epimer separation; LC-MS/MS; Metabolites; PTAD; Vitamin D

DOI:  https://doi.org/10.1016/j.jpba.2023.115522
Clin Chim Acta. 2023 Jun 16. pii: S0009-8981(23)00251-6. [Epub ahead of print] 117449

Sensitive LC-MS/MS method for the temporal profiling of bile acids, fatty acids and branched-chain alpha-keto acids in maternal plasma during pregnancy and cord blood plasma at delivery.

Daniel Zhi Wei Ng, Sean Xian Yu Lee, Delicia Shu Qin Ooi, Le Duc Huy Ta, Gaik Chin Yap, Carina Jing Xuan Tay, Chiung-Hui Huang, Elizabeth Huiwen Tham, Evelyn Xiu Ling Loo, Lynette P C Shek, Anne Goh, Hugo P S Van Bever, Oon Hoe Teoh, Yung Seng Lee, Fabian Yap, Kok Hian Tan, Yap-Seng Chong, Shiao Yng Chan, Johan Gunnar Eriksson, Keith M Godfrey, Bee Wah Lee, Eric Chun Yong Chan.

   BACKGROUND AND AIMS: There are significant changes to the maternal inflammatory profile across pregnancy. Recent studies suggest that perturbations in maternal gut microbial and dietary-derived plasma metabolites over the course of pregnancy mediate inflammation through a complex interplay of immunomodulatory effects. Despite this body of evidence, there is currently no analytical method that is suitable for the simultaneous profiling of these metabolites within human plasma.
MATERIALS AND METHODS: We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the high-throughput analysis of these metabolites in human plasma without derivatization. Plasma samples were processed using liquid-liquid extraction method with varying proportions of methyl tert-butyl ether, methanol, and water in a 3:10:2.5 ratio to reduce matrix effects.
RESULTS: LC-MS/MS detection was sufficiently sensitive to quantify these gut microbial and dietary-derived metabolites at physiological concentrations and linear calibration curves with r2>0.99 were obtained. Recovery was consistent across concentration levels. Stability experiments confirmed that up to 160 samples could be analyzed within a single batch. The method was validated and applied to analyse maternal plasma during the first and third trimester and cord blood plasma of 5 mothers.
CONCLUSION: This study validated a straightforward and sensitive LC/MS-MS method for the simultaneous quantitation of gut microbial and dietary-derived metabolites in human plasma within 9 minutes without prior sample derivatization.

Keywords:  Bile acid; Branch chain alpha keto acid; Fatty acid; Liquid chromatography tandem mass spectrometry; Liquid-liquid extraction; Short chain fatty acid

DOI:  https://doi.org/10.1016/j.cca.2023.117449
Metabolomics. 2023 Jun 23. 19(7): 61

Capillary electrophoresis-mass spectrometry as a tool for Caenorhabditis elegans metabolomics research.

Liesa Salzer, Philippe Schmitt-Kopplin, Michael Witting.

   INTRODUCTION: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes.
OBJECTIVE: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome.
METHOD: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument.
RESULTS: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites.
CONCLUSION: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.

Keywords:  CE-MS; Caenorhabditis elegans; Capillary electrophoresis; HILIC-MS; Mass spectrometry; Metabolomics; daf-2

DOI:  https://doi.org/10.1007/s11306-023-02025-7
BMC Bioinformatics. 2023 Jun 17. 24(1): 259

A MATLAB-based app to improve LC-MS/MS data analysis for N-linked glycan peak identification.

Ashna Dhingra, Zayla Schaeffer, Natalia I Majewska Nepomuceno, Jennifer Au, Joomi Ahn.

   BACKGROUND: Glycosylation is an important modification to proteins that plays a significant role in biological processes. Glycan structures are characterized by liquid chromatography (LC) combined with mass spectrometry (MS), but data interpretation of LC/MS and MS/MS data can be time-consuming and arduous when analyzed manually. Most of glycan analysis requires dedicated glycobioinformatics tools to process MS data, identify glycan structure, and display the results. However, software tools currently available are either too costly or heavily focused on academic applications, limiting their use within the biopharmaceutical industry for implementing the standardized LC/MS glycan analysis in high-throughput manner. Additionally, few tools provide the capability to generate report-ready annotated MS/MS glycan spectra.
RESULTS: Here, we present a MATLAB-based app, GlyKAn AZ, which can automate data processing, glycan identification, and customizable result displays in a streamlined workflow. MS1 and MS2 mass search algorithms along with glycan databases were developed to confirm the fluorescent labeled N-linked glycan species based on accurate mass. A user-friendly graphical user interface (GUI) streamlines the data analysis process, making it easy to implement the software tool in biopharmaceutical analytical laboratories. The databases provided with the app can be expanded through the Fragment Generator functionality which automatically identifies fragmentation patterns for new glycans. The GlyKAn AZ app can automatically annotate the MS/MS spectra, yet this data display feature remains flexible and customizable by users, saving analysts' time in generating individual report-ready spectra figures. This app accepts both OrbiTrap and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS data and was successfully validated by identifying all glycan species that were previously identified manually.
CONCLUSIONS: The GlyKAn AZ app was developed to expedite glycan analysis while maintaining a high level of accuracy in positive identifications. The app's customizable user inputs, polished figures and tables, and unique calculated outputs set it apart from similar software and greatly improve the current manual analysis workflow. Overall, this app serves as a tool for streamlining glycan identification for both academic and industrial needs.

Keywords:  Glycans; Glycosylation; Liquid chromatography; MATLAB; Matrix-assisted laser desorption/ionization; Tandem mass spectrometry

DOI:  https://doi.org/10.1186/s12859-023-05346-5
J Chromatogr A. 2023 Jun 19. pii: S0021-9673(23)00400-4. [Epub ahead of print]1705 464174

Vacuum differential mobility spectrometry combined with column-switching liquid chromatography- mass spectrometry for the analysis of pyrrolizidine alkaloids in tea samples.

Maria Fernanda Cifuentes Girard, Patrick Knight, Gérard Hopfgartner.

  The benefit of combining liquid chromatography (LC), supercritical fluid chromatography (SFC) and vacuum Differential Mobility Spectrometry - Mass Spectrometry (vDMS-MS) was investigated for the analysis of fourteen diastereomeric pyrrolizidine alkaloids (PA); intermedine, echinatine, lycopsamine, indicine, intermedine-N-oxide, echinatine-N-oxide, indicine-N-oxide, lycopsamine-N-oxide, senecivernine, senecionine, jacobine, senecivernine-N-oxide, senecionine-N-oxide, retrorsine. The mobile phase composition (15-100% MeOH and ACN), flow rate (8-100 µL/min), vDMS cell pressure, and F value showed an effect on the mobility behavior of the analytes. At 15% MeOH with a flow rate of 100 µL/min and 33 mbar vDMS pressure, 8 out 14 PA could be partially or totally separated by vDMS-MS. As well as providing an additional separation dimension vDMS improved the selectivity and a 5-minute assay method was developed for the quantification of 10 out of 14 single diastereomeric PA in tea samples, using a short LC column-switching and hyphenated to vDMS-MS in the selected ion monitoring mode. The performance of the method was found to be comparable with a 12-minute standard LC-MS/MS method using detection in the selected reaction monitoring mode. Additionally, the combination of vDMS and SFC-MS was investigated and suggests that the mixture of CO2/MeOH influences the CV shifting of the PA to more negative compensation voltage, and the signal-to-noise ratio is improved by a factor of three compared to SFC-MS without vDMS.

Keywords:  Column switching LC-MS; Differential mobility spectrometry; Pyrrolizidine alkaloids; Supercritical fluid Chromatography-MS; Tea

DOI:  https://doi.org/10.1016/j.chroma.2023.464174
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jun 15. pii: S1570-0232(23)00196-4. [Epub ahead of print]1226 123786

A sensitive and rapid LC-MS/MS assay for quantitation of free payload Aur0101 from antibody drug conjugate (ADC) PYX-201 in human plasma.

Feng Yin, Diana Adhikari, Yan Li, Devan Turner, M Shane Woolf, Diane Lebarbenchon, Eric Ma, William Mylott, Elizabeth Shaheen, Shawn Harriman, Jan Pinkas.

  PYX-201 is an investigational antibody drug conjugate (ADC) with an engineered, fully human IgG1 antibody, a cleavable chemical linker, and a toxin (Aur0101) with an average drug-antibody ratio (DAR) of ∼ 4. A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated to determine the presence in human plasma, of free payload Aur0101 from PYX-201 to assess drug safety and efficacy. Aur0101 and its deuterated internal standard (IS), Aur0101_d8, were extracted from 25 µL of human plasma using a solid liquid extraction (SLE) method. Chromatographic analysis was carried out on a Waters Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7 µm, 130 A) column. Quantitation of free Aur0101 was conducted on a Sciex triple quadrupole mass spectrometer API 6500 + using multiple reaction monitoring (MRM) mode via positive electrospray ionization. The calibration curve was linear over the concentration range of 25.0 to 12,500 pg/mL with correlation coefficient, r2 ≥ 0.9988. The intra-assay %RE was between -4.3% to 14.3% with % CV was ≤ 6.2%. The inter-assay %RE was between -0.2% to 9.5% with % CV was ≤ 6.1%. The average analyte recovery was 89.7% and the average IS recovery was 88.7%. Aur0101 was found to be stable in human plasma and human whole blood under various tested conditions with and without the presence of PYX-201. To our knowledge, this is the first published fully validated assay for free, unconjugated Aur0101 in any matrix, from any species. This assay has been successfully applied to clinical sample analysis to support clinical studies.

Keywords:  ADC; Aur0101; Human plasma; LC-MS/MS; Oncology; PYX-201; Validation; extra domain B fibronectin

DOI:  https://doi.org/10.1016/j.jchromb.2023.123786
Biomed Chromatogr. 2023 Jun 22. e5689

Simultaneous quantitation of favipiravir and its hydroxide metabolite in human plasma and hamster matrices using a UPLC-MS/MS method.

Cesar P Bekegnran, Jean-Selim Driouich, Judith Breuer, Karine Barthelemy, Madeleine Giocanti, Xavier de Lamballerie, Alexandra Y Kreins, Antoine Nougairede, Caroline Solas.

  Favipiravir, a broad-spectrum RNA-dependent RNA polymerase inhibitor, is currently being evaluated in preclinical and clinical studies for the treatment of various infectious diseases including COVID-19. We developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of favipiravir and its hydroxide metabolite (M1), in human and hamster biological matrices. Analytes were separated on an Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) after a simple protein precipitation with acetonitrile. The mobile phase consisted of water and methanol, each containing 0.05% formic acid. Experiments were performed using electrospray ionization in the positive and negative ion mode, with protonated molecules used as the precursor ion and a total run time of 6 min. The MS/MS response was linear over the concentration ranges from 0.5-100 μg/ml for favipiravir and 0.25-30 μg/ml for M1. Intra- and inter-day accuracy and precision were within the recommended limits of the European Medicines Agency guidelines. No significant matrix effect was observed, and the method was successfully applied to inform favipiravir dose adjustments in six immunocompromised children with severe RNA viral infections. In conclusion, the UPLC-MS/MS assay is suitable for quantification of favipiravir over a wide range of dosing regimens, and can easily be adapted to other matrices and species.

Keywords:  UPLC-MS/MS; favipiravir; lung homogenate; metabolite M1; plasma

DOI:  https://doi.org/10.1002/bmc.5689
J Pharm Biomed Anal. 2023 Jun 17. pii: S0731-7085(23)00306-0. [Epub ahead of print]234 115537

Precolumn derivatization LC-MS/MS method to simultaneous quantitation of 10 monosaccharides in rat plasma.

Mengxue Li, Ping Li, Yuting Ji, Yihua Tian, Huiying Zeng, Xiaoyan Chen.

  Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100-5.00 μg/mL for rhamnose, 0.0500-25.0 μg/mL for fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine, 1.00-500 μg/mL for galactose, 10.0-5000 μg/mL for mannose, and 50.0-25,000 μg/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury.

Keywords:  1-Phenyl-3-methyl-5-pyrazolone (PMP); Biomarker; Derivatization; LC-MS/MS; Monosaccharides

DOI:  https://doi.org/10.1016/j.jpba.2023.115537
Anal Bioanal Chem. 2023 Jun 17.

Desorption electrospray ionization and matrix-assisted laser desorption/ionization as imaging approaches for biological samples analysis.

Lanaia Ítala Louzeiro Maciel, Ricardo Alves Bernardo, Rafael Oliveira Martins, Almir Custodio Batista Junior, João Victor Ataíde Oliveira, Andréa Rodrigues Chaves, Boniek Gontijo Vaz.

  The imaging of biological tissues can offer valuable information about the sample composition, which improves the understanding of analyte distribution in such complex samples. Different approaches using mass spectrometry imaging (MSI), also known as imaging mass spectrometry (IMS), enabled the visualization of the distribution of numerous metabolites, drugs, lipids, and glycans in biological samples. The high sensitivity and multiple analyte evaluation/visualization in a single sample provided by MSI methods lead to various advantages and overcome drawbacks of classical microscopy techniques. In this context, the application of MSI methods, such as desorption electrospray ionization-MSI (DESI-MSI) and matrix-assisted laser desorption/ionization-MSI (MALDI-MSI), has significantly contributed to this field. This review discusses the evaluation of exogenous and endogenous molecules in biological samples using DESI and MALDI imaging. It offers rare technical insights not commonly found in the literature (scanning speed and geometric parameters), making it a comprehensive guide for applying these techniques step-by-step. Furthermore, we provide an in-depth discussion of recent research findings on using these methods to study biological tissues.

Keywords:  Biological samples; Desorption electrospray ionization; Mass spectrometry imaging; Matrix-assisted laser desorption/ionization

DOI:  https://doi.org/10.1007/s00216-023-04783-8
Crit Rev Anal Chem. 2023 Jun 22. 1-15

A Critical Review on Chemical Speciation of Chlorine-Produced Oxidants in Seawater. Part 3: Chromatographic- and Mass Spectrometric-Based Methodologies.

Adrien Roumiguières, Stéphane Bouchonnet, Said Kinani.

  Chlorination of seawater forms a range of secondary oxidative species - collectively called "chlorine-produced oxidants" (CPOs) - having different biocidal, environmental and ecotoxicological properties. The chemical speciation of these compounds is an important step in attempts to assess the effectiveness of chlorination and the potential impacts of its releases. However, comprehensive determination of CPOs represents a significant analytical challenge for many reasons, including the following: CPO species are numerous, highly reactive, with short-lifetimes, difficult to isolate and generally present at low concentrations in a complex salt matrix. Literature review reveals the development of a wide variety of analytical approaches for analysis of CPOs, either collectively via group parameters or individually. A first category of these approaches was the subject of article II (also including sampling and sample preparation) of a trilogy devoted to the chemical speciation of CPOs in seawater. In this third article - which closes the trilogy - emphasis is placed on chromatographic- and mass spectrometric-based approaches. It reviews more than 80 methods, reported from 1981 to date, and thoroughly discusses their principles and performances. Methodologies involving chemical derivatization of CPOs prior to their analysis by gas or liquid chromatography coupled to mass spectrometry provide the best sensitivities, achieving sub-ppb detection limits for species for which suitable derivatization reagents are available. Online mass spectrometry approaches are attracting increasing interest for their ability to analyze multiple CPO species in real time without extensive sample preparation steps, reaching detection limits of about ppb for less polar oxidants. At the current state of metrological development, neither the methodologies based on chromatography nor those based on online mass spectrometry allow complete speciation of CPOs. Future trends and major challenges related to these approaches are discussed.

Keywords:  Chlorination; chlorine-produced oxidants (CPOs); gas chromatography; liquid chromatography; membrane introduction (or inlet) mass spectrometry (MIMS); seawater

DOI:  https://doi.org/10.1080/10408347.2023.2220129
Nat Commun. 2023 Jun 22. 14(1): 3722

Ultra-fast and accurate electron ionization mass spectrum matching for compound identification with million-scale in-silico library.

Qiong Yang, Hongchao Ji, Zhenbo Xu, Yiming Li, Pingshan Wang, Jinyu Sun, Xiaqiong Fan, Hailiang Zhang, Hongmei Lu, Zhimin Zhang.

Spectrum matching is the most common method for compound identification in mass spectrometry (MS). However, some challenges limit its efficiency, including the coverage of spectral libraries, the accuracy, and the speed of matching. In this study, a million-scale in-silico EI-MS library is established. Furthermore, an ultra-fast and accurate spectrum matching (FastEI) method is proposed to substantially improve accuracy using Word2vec spectral embedding and boost the speed using the hierarchical navigable small-world graph (HNSW). It achieves 80.4% recall@10 accuracy (88.3% with 5 Da mass filter) with a speedup of two orders of magnitude compared with the weighted cosine similarity method (WCS). When FastEI is applied to identify the molecules beyond NIST 2017 library, it achieves 50% recall@1 accuracy. FastEI is packaged as a standalone and user-friendly software for common users with limited computational backgrounds. Overall, FastEI combined with a million-scale in-silico library facilitates compound identification as an accurate and ultra-fast tool.

DOI: https://doi.org/10.1038/s41467-023-39279-7
Anal Chem. 2023 Jun 21.

Volume-Corrected Free Energy as a New Criterion for Structural Elucidation in Chemical-Tagging-Based Metabolomics.

Hua-Ming Xiao, Xiao-Hui Peng, Di Rao, Shuai Zhao, Dilshad Hussain, Jian-Li Chen, Dan Luo, Dan Wang, Xin Lv, Xian Wang, Fang Wei, Hong Chen.

Chemical tagging via possible derivatization reagents alters metabolites' retention times, leading to different retention behavior during liquid chromatography-mass spectrometry (LC-MS) analysis. Incorporation of the retention time dimension can dramatically reduce false-positive structural elucidation in chemical-tagging-based metabolomics. However, few studies predict the retention times of chemically labeled metabolites, especially requiring a simple, easy-to-access, accurate, and universal predictor or descriptor. This pilot study demonstrates the application of volume-corrected free energy (VFE) calculation and region mapping as a new criterion to describe the retention time for structure elucidation in chemical-tagging-based metabolomics. The universality of VFE calculation is first evaluated with four different types of submetabolomes including hydroxyl-group-, carbonyl-group-, carboxylic-group-, and amino-group-containing compounds and oxylipins with similar chemical structures and complex isomers on reverse-phase LC. Results indicate a good correlation (r > 0.85) between VFE values and their corresponding retention times using different technicians, instruments, and chromatographic columns, describing retention behavior in reverse-phase LC. Finally, the VFE region mapping is described for identifying 1-pentadecanol from aged camellia seed oil using three proposed steps, including public database searching, VFE region mapping for its 12 isomers, and chemical standard matching. The possibility of VFE calculation of nonderivatized compounds in retention time prediction is also investigated, demonstrating its effectiveness on retention times with different influence factors.

DOI: https://doi.org/10.1021/acs.analchem.3c00722
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jun 14. pii: S1570-0232(23)00200-3. [Epub ahead of print]1226 123790

A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs.

Jianli Zhang, Jianghai Lu, Yinong Zhang, Yan Wang.

   OBJECTIVES: To establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol & S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs.
METHODS: A LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid-liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated.
RESULTS: R-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5-500 ng/g. Accuracies ranged from -11.9-13.0% for R-(-)-clenbuterol and -10.2-13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6-5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1.
CONCLUSIONS: The analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.

Keywords:  Animal tissue; Doping control; Food safety; LC-MS/MS; R-(-)-clenbuterol; S-(+)-clenbuterol

DOI:  https://doi.org/10.1016/j.jchromb.2023.123790
Anal Bioanal Chem. 2023 Jun 20.

Fast profiling of primary, secondary, conjugated, and sulfated bile acids in human urine and murine feces samples.

Victoria Ramos-Garcia, Isabel Ten-Doménech, Máximo Vento, Clara Bullich-Vilarrubias, Marina Romaní-Pérez, Yolanda Sanz, Angelica Nobili, Marika Falcone, Marina Di Stefano, Guillermo Quintás, Julia Kuligowski.

  Bile acids (BAs) are a complex class of metabolites that have been described as specific biomarkers of gut microbiota activity. The development of analytical methods allowing the quantification of an ample spectrum of BAs in different biological matrices is needed to enable a wider implementation of BAs as complementary measures in studies investigating the functional role of the gut microbiota. This work presents results from the validation of a targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 28 BAs and six sulfated BAs, covering primary, secondary, and conjugated BAs. The analysis of 73 urine and 20 feces samples was used to test the applicability of the method. Concentrations of BAs in human urine and murine feces were reported, ranging from 0.5 to 50 nmol/g creatinine and from 0.012 to 332 nmol/g, respectively. Seventy-nine percent of BAs present in human urine samples corresponded to secondary conjugated BAs, while 69% of BAs present in murine feces corresponded to primary conjugated BAs. Glycocholic acid sulfate (GCA-S) was the most abundant BA in human urine samples, while taurolithocholic acid was the lowest concentrated compound detected. In murine feces, the most abundant BAs were α-murocholic, deoxycholic, dehydrocholic, and β-murocholic acids, while GCA-S was the lowest concentrated BA. The presented method is a non-invasive approach for the simultaneous assessment of BAs and sulfated BAs in urine and feces samples, and the results will serve as a knowledge base for future translational studies focusing on the role of the microbiota in health.

Keywords:  Bile acids; Human urine; Microbiota; Murine feces

DOI:  https://doi.org/10.1007/s00216-023-04802-8
Drug Test Anal. 2023 Jun 21.

Determination of cannabinoids in 50 μL whole blood samples by online extraction using turbulent flow chromatography and LC-HRAM-Orbitrap-MS: Application on driving under the influence of drugs cases.

Flavio Zancanaro, Gianpaola Tedeschi, Luca Zamengo, Samuela Frasson, Giampietro Frison.

  The analysis of cannabinoids in whole blood is usually done by traditional mass spectrometry (MS) techniques, after offline cleanup or derivatization steps which can be lengthy, laborious, and expensive. We present a simple, fast, highly specific, and sensitive method for the determination of Δ9 -tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-hydroxy-Δ9 -tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in 50 μL whole blood samples. After the addition of deuterated internal standards (IS) and a simple protein precipitation step, an online extraction of sample supernatants using turbulent flow chromatography (TurboFlow-Thermo Scientific) was carried out. Analytes were separated on a C18 analytical column and detected by LC-HRAM-Orbitrap-MS using a Thermo Scientific Q Exactive Focus MS system. MS detection was performed in polarity switching and selected ion monitoring (SIM) modes using five specific acquisition windows, at a resolution of 70,000 (FWHM). Total run time was about 10 min including preanalytical steps. Method validation was carried out by determining limit of detection (LOD), lower limit of quantitation (LLOQ), linearity range, analytical accuracy, intra-assay and interassay precision, carry-over, matrix effect, extraction recovery, and selectivity, for all analytes. Measurement uncertainties were also evaluated, and a decision rule was set with confidence for forensic purposes. The method may become suitable for clinical and forensic toxicology applications, taking advantage of the small matrix volume required, the simple and cost-effective sample preparation procedure, and the fast analytical run time. Performances were monitored over a long-term period and tested on 7620 driving under the influence of drugs (DUID) samples, including 641 positive samples.

Keywords:  DUID; Orbitrap™; cannabinoids; high-resolution mass spectrometry; whole blood

DOI:  https://doi.org/10.1002/dta.3532
Front Plant Sci. 2023 ;14 1178239

Systematic comparison of local approaches for isotopically nonstationary metabolic flux analysis.

Sebastian Huß, Zoran Nikoloski.

  Quantification of reaction fluxes of metabolic networks can help us understand how the integration of different metabolic pathways determine cellular functions. Yet, intracellular fluxes cannot be measured directly but are estimated with metabolic flux analysis (MFA) that relies on the patterns of isotope labeling of metabolites in the network. For metabolic systems, typical for plants, where all potentially labeled atoms effectively have only one source atom pool, only isotopically nonstationary MFA can provide information about intracellular fluxes. There are several global approaches that implement MFA for an entire metabolic network and estimate, at once, a steady-state flux distribution for all reactions with identifiable fluxes in the network. In contrast, local approaches deal with estimation of fluxes for a subset of reactions, with smaller data demand for flux estimation. Here we present a systematic comparative review and benchmarking of the existing local approaches for isotopically nonstationary MFA. The comparison is conducted with respect to the required data and underlying computational problems solved on a synthetic network example. Furthermore, we benchmark the performance of these approaches in estimating fluxes for a subset of reactions using data obtained from the simulation of nitrogen fluxes in the Arabidopsis thaliana core metabolism. The findings pinpoint practical aspects that need to be considered when applying local approaches for flux estimation in large-scale plant metabolic networks.

Keywords:  comparison; flux estimation; isotopically nonstationary; local; metabolic flux analysis

DOI:  https://doi.org/10.3389/fpls.2023.1178239
Front Mol Biosci. 2023 ;10 1125582

Quantitative plasma profiling by 1H NMR-based metabolomics: impact of sample treatment.

Francisco Madrid-Gambin, Sergio Oller, Santiago Marco, Óscar J Pozo, Cristina Andres-Lacueva, Rafael Llorach.

  Introduction: There is evidence that sample treatment of blood-based biosamples may affect integral signals in nuclear magnetic resonance-based metabolomics. The presence of macromolecules in plasma/serum samples makes investigating low-molecular-weight metabolites challenging. It is particularly relevant in the targeted approach, in which absolute concentrations of selected metabolites are often quantified based on the area of integral signals. Since there are a few treatments of plasma/serum samples for quantitative analysis without a universally accepted method, this topic remains of interest for future research. Methods: In this work, targeted metabolomic profiling of 43 metabolites was performed on pooled plasma to compare four methodologies consisting of Carr-Purcell-Meiboom-Gill (CPMG) editing, ultrafiltration, protein precipitation with methanol, and glycerophospholipid solid-phase extraction (g-SPE) for phospholipid removal; prior to NMR metabolomics analysis. The effect of the sample treatments on the metabolite concentrations was evaluated using a permutation test of multiclass and pairwise Fisher scores. Results: Results showed that methanol precipitation and ultrafiltration had a higher number of metabolites with coefficient of variation (CV) values above 20%. G-SPE and CPMG editing demonstrated better precision for most of the metabolites analyzed. However, differential quantification performance between procedures were metabolite-dependent. For example, pairwise comparisons showed that methanol precipitation and CPMG editing were suitable for quantifying citrate, while g-SPE showed better results for 2-hydroxybutyrate and tryptophan. Discussion: There are alterations in the absolute concentration of various metabolites that are dependent on the procedure. Considering these alterations is essential before proceeding with the quantification of treatment-sensitive metabolites in biological samples for improving biomarker discovery and biological interpretations. The study demonstrated that g-SPE and CPMG editing are effective methods for removing proteins and phospholipids from plasma samples for quantitative NMR analysis of metabolites. However, careful consideration should be given to the specific metabolites of interest and their susceptibility to the sample treatment procedures. These findings contribute to the development of optimized sample preparation protocols for metabolomics studies using NMR spectroscopy.

Keywords:  metabolomics; nuclear magnetic resonance; plasma; quantification; quantitative analysis; sample treatment

DOI:  https://doi.org/10.3389/fmolb.2023.1125582