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Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research
Ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC–HRMS) variants currently represent the best tools to tackle the challenges of complexity and lack of comprehensive coverage of the metabolome. UHPLC offers flexible and efficient separation coupled with high-sensitivity detection via HRMS, allowing for the detection and identification of a broad range of metabolites. Here we discuss current common strategies for UHPLC–HRMS-based metabolomics, with a focus on expanding metabolome coverage.This Review surveys ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC–HRMS), a highly sensitive, high-throughput technique that is used for analyzing a broad range of metabolites.
Journal Article
Current trends in supercritical fluid chromatography
Supercritical fluid chromatography (SFC), which employs pressurized carbon dioxide as the major component of the mobile phase, has been known for several decades but has faced a significant resurgence of interest in the recent years, thanks to the development of modern instruments to comply with current expectations in terms of robustness and sensitivity. This review is focused on the recent literature, specifically since the introduction of modern systems but in relation to older literature, to identify the changing trends in application domains. Typically, natural products, bioanalysis, food science, and environmental analyses are all strongly increasing. Together with reduced extra-column volumes in the instruments, the advent of sub-2-μm particles and superficially porous particles in the stationary phases is favoring ultra-high-performance SFC (UHPSFC) allowing for improved resolution and faster analyses, but without the constraints of viscous liquids encountered in ultra-high-performance liquid chromatography (UHPLC). Hyphenation to mass spectrometry is also more frequent and opened the way to new application domains, and raises different issues from liquid chromatography mobile phases, especially due to decompression of carbon dioxide. It is also shown that the frontiers between SFC and HPLC are fading, as switching from one method to the other, even within the course of a single analysis, is facilitated my modern instruments. The present review is not intended to be exhaustive but rather giving a snapshot of recent trends in supercritical fluid chromatography, based on the observation of about 500 papers published in English-written peer-reviewed journals from 2014 to 2018.
Journal Article
Simultaneous determination of perfluoroalkyl substances and bile acids in human serum using ultra-high-performance liquid chromatography–tandem mass spectrometry
There is evidence of a positive association between per- and polyfluoroalkyl substances (PFASs) and cholesterol levels in human plasma, which may be due to common reabsorption of PFASs and bile acids (BAs) in the gut. Here we report development and validation of a method that allows simultaneous, quantitative determination of PFASs and BAs in plasma, using 150 μL or 20 μL of sample. The method involves protein precipitation using 96-well plates. The instrumental analysis was performed with ultra-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS), using reverse-phase chromatography, with the ion source operated in negative electrospray mode. The mass spectrometry analysis was carried out using multiple reaction monitoring mode. The method proved to be sensitive, robust, and with sufficient linear range to allow reliable determination of both PFASs and BAs. The method detection limits were between 0.01 and 0.06 ng mL−1 for PFASs and between 0.002 and 0.152 ng mL−1 for BAs, with the exception of glycochenodeoxycholic acid (0.56 ng mL−1). The PFAS measured showed excellent agreement with certified plasma PFAS concentrations in NIST SRM 1957 reference serum. The method was tested on serum samples from 20 healthy individuals. In this proof-of-concept study, we identified significant associations between plasma PFAS and BA levels, which suggests that PFAS may alter the synthesis and/or uptake of BAs.
Journal Article
Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography–mass spectrometry
Untargeted methods are typically used in the detection and discovery of small organic compounds in metabolomics research, and ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS) is one of the most commonly used platforms for untargeted metabolomics. Although they are non-biased and have high coverage, untargeted approaches suffer from unsatisfying repeatability and a requirement for complex data processing. Targeted metabolomics based on triple-quadrupole mass spectrometry (TQMS) could be a complementary tool because of its high sensitivity, high specificity and excellent quantification ability. However, it is usually applicable to known compounds: compounds whose identities are known and/or are expected to be present in the analyzed samples. Pseudotargeted metabolomics merges the advantages of untargeted and targeted metabolomics and can act as an alternative to the untargeted method. Here, we describe a detailed protocol of pseudotargeted metabolomics using UHPLC-TQMS. An in-depth, untargeted metabolomics experiment involving multiple UHPLC-HRMS runs with MS at different collision energies (both positive and negative) is performed using a mixture obtained using small amounts of the analyzed samples. XCMS, CAMERA and Multiple Reaction Monitoring (MRM)-Ion Pair Finder are used to find and annotate peaks and choose transitions that will be used to analyze the real samples. A set of internal standards is used to correct for variations in retention time. High coverage and high-performance quantitative analysis can be realized. The entire protocol takes ~5 d to complete and enables the simultaneously semiquantitative analysis of 800–1,300 metabolites.
In pseudotargeted metabolomics, transitions used for multiple-reaction monitoring are chosen from mass spectrometry data obtained using mixtures of real samples. Semiquantitative information is obtained without knowing the identity of the compounds.
Journal Article
Analysis of phenolic compounds in different parts of pomegranate (Punica granatum) fruit by HPLC-PDA-ESI/MS and evaluation of their antioxidant activity: application to different Italian varieties
The analysis of pomegranate phenolic compounds belonging to different classes in different fruit parts was performed by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detection. Two different separation methods were optimized for the analysis of anthocyanins and hydrolyzable tannins along with phenolic acids and flavonoids. Two C18 columns, core–shell and fully porous particle stationary phases, were used. The parameters for separation of phenolic compounds were optimized considering chromatographic resolution and analysis time. Thirty-five phenolic compounds were found, and 28 of them were tentatively identified as belonging to four different phenolic compound classes; namely, anthocyanins, phenolic acids, hydrolyzable tannins, and flavonoids. Quantitative analysis was performed with a mixture of nine phenolic compounds belonging to phenolic compound classes representative of pomegranate. The method was then fully validated in terms of retention time precision, expressed as the relative standard deviation, limit of detection, limit of quantification, and linearity range. Phenolic compounds were analyzed directly in pomegranate juice, and after solvent extraction with a mixture of water and methanol with a small percentage of acid in peel and pulp samples. The accuracy of the extraction method was also assessed, and satisfactory values were obtained. Finally, the method was used to study identified analytes in pomegranate juice, peel, and pulp of six different Italian varieties and one international variety. Differences in phenolic compound profiles among the different pomegranate parts were observed. Pomegranate peel samples showed a high concentration of phenolic compounds, ellagitannins being the most abundant ones, with respect to pulp and juice samples for each variety. With the same samples, total phenols and antioxidant activity were evaluated through colorimetric assays, and the results were correlated among them.
Journal Article
MetaboAnalystR 4.0: a unified LC-MS workflow for global metabolomics
The wide applications of liquid chromatography - mass spectrometry (LC-MS) in untargeted metabolomics demand an easy-to-use, comprehensive computational workflow to support efficient and reproducible data analysis. However, current tools were primarily developed to perform specific tasks in LC-MS based metabolomics data analysis. Here we introduce MetaboAnalystR 4.0 as a streamlined pipeline covering raw spectra processing, compound identification, statistical analysis, and functional interpretation. The key features of MetaboAnalystR 4.0 includes an auto-optimized feature detection and quantification algorithm for LC-MS1 spectra processing, efficient MS2 spectra deconvolution and compound identification for data-dependent or data-independent acquisition, and more accurate functional interpretation through integrated spectral annotation. Comprehensive validation studies using LC-MS1 and MS2 spectra obtained from standards mixtures, dilution series and clinical metabolomics samples have shown its excellent performance across a wide range of common tasks such as peak picking, spectral deconvolution, and compound identification with good computing efficiency. Together with its existing statistical analysis utilities, MetaboAnalystR 4.0 represents a significant step toward a unified, end-to-end workflow for LC-MS based global metabolomics in the open-source R environment.
Several bottlenecks exist in metabolomics data analysis. Here, the authors present MetaboAnalystR 4.0 as a unified workflow for LC-MS untargeted metabolomics. It highlights significant improvements in LC-MS2 spectral processing and functional analysis, providing an end-to-end computational pipeline.
Journal Article
Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices
Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20–100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples.
Journal Article
Tissue Distribution of 5-Hydroxymethylcytosine and Search for Active Demethylation Intermediates
5-Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5-formylcytosine (fC), 5-carboxylcytosine (caC), and 5-hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.
Journal Article
Autonomous mobile robots for exploratory synthetic chemistry
Autonomous laboratories can accelerate discoveries in chemical synthesis, but this requires automated measurements coupled with reliable decision-making
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. Most autonomous laboratories involve bespoke automated equipment
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, and reaction outcomes are often assessed using a single, hard-wired characterization technique
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. Any decision-making algorithms
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must then operate using this narrow range of characterization data
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,
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. By contrast, manual experiments tend to draw on a wider range of instruments to characterize reaction products, and decisions are rarely taken based on one measurement alone. Here we show that a synthesis laboratory can be integrated into an autonomous laboratory by using mobile robots
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that operate equipment and make decisions in a human-like way. Our modular workflow combines mobile robots, an automated synthesis platform, a liquid chromatography–mass spectrometer and a benchtop nuclear magnetic resonance spectrometer. This allows robots to share existing laboratory equipment with human researchers without monopolizing it or requiring extensive redesign. A heuristic decision-maker processes the orthogonal measurement data, selecting successful reactions to take forward and automatically checking the reproducibility of any screening hits. We exemplify this approach in the three areas of structural diversification chemistry, supramolecular host–guest chemistry and photochemical synthesis. This strategy is particularly suited to exploratory chemistry that can yield multiple potential products, as for supramolecular assemblies, where we also extend the method to an autonomous function assay by evaluating host–guest binding properties.
A modular autonomous platform for general exploratory synthetic chemistry uses mobile robots to integrate an automated synthesis platform and two analysis platforms.
Journal Article