bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒07‒12
sixteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Anal Chem. 2020 Jul 10.
      Data-independent acquisition mass spectrometry (DIA-MS) is essential for information-rich spectral annotations in untargeted metabolomics. However, the acquired MS2 spectra are highly complex, posing significant annotation challenges. We have developed a correlation-based deconvolution (CorrDec) method that uses ion abundance correlations in multi-sample studies using DIA-MS as an update of our MS-DIAL software. CorrDec is based on the assumption that peak intensities of precursor and fragment ions correlate across samples, and exploits this quantitative information to deconvolute complex DIA spectra. CorrDec clearly improved deconvolution of the original MS-DIAL deconvolution method (MS2Dec) in a dilution series of chemical standards and a 224-sample urinary metabolomics study. The primary advantage of CorrDec over MS2Dec is the ability to discriminate co-eluting low-abundance compounds. CorrDec requires the measurement of multiple samples to successfully deconvolute DIA spectra, and our randomized assessment demonstrated that CorrDec can contribute to studies with as few as 10 unique samples. The presented methodology improves compound annotation and identification in multi-sample studies and will be useful for applications in large cohort studies.
  2. Rapid Commun Mass Spectrom. 2020 Jul 06. e8876
      RATIONALE: Electrospray ionization mass spectrometry (ESI-MS) in conjunction with liquid chromatography (LC) can provide accurate quantitative data, but it is not well-suited for rapid screening (RS) of analytes incurred into complex matrices. This study was designed to determine the usefulness of ESI for rapid detection and quantitation of veterinary drugs from complex biological matrices under near real-time conditions.METHODS: Nine veterinary drugs or metabolites, clenbuterol, erythromycin, flunixin, 5-hydroxyflunixin, meloxicam, ractopamine, salbutamol, tylosin, and zilpaterol present in cow urine, sheep urine, sheep tissues (kidney, muscle, liver and lung) or pig kidney were simultaneously analyzed. A simple sample clean-up procedure, which included dilution with 10% sodium carbonate followed by extraction with ethyl acetate, was used. For tissues, an additional pre-extraction with hexane was performed to remove fat prior to MS analysis. Samples were introduced into the mass spectrometer through the LC autosampler, but no chromatographic separation was employed. A Sciex 5600+ triple time-of-flight mass spectrometer with a dual-spray source, interfaced with a Shimadzu Nexera LC system was used. Samples were analyzed in positive ion mode.
    RESULTS: Sample extraction times were typically 10 - 30 min or less and instrumental analysis time was 1 min/sample. Regression coefficients of matrix-matched standard curves across all compounds ranged from 0.9701 - 0.9999 in urine (cow and sheep) and tissues (sheep kidney, liver, lung, muscle and pig kidney). Limits of detection ranged from 0.11 to 2.03 ng/mL across analyte in urine and 0.11 to 8.00 ng/g across tissues. Correlations between RS-ESI-MS and LC/MS/MS results were 0.956 to 0.998 for incurred residues of flunixin in cow urine, ractopamine in pig kidney, and zilpaterol in sheep urine.
    CONCLUSIONS: RS-ESI-MS provided rapid, sensitive, and accurate analyses of nine veterinary drugs from complex matrices with very little sample preparation and produced quantitative data akin to LC/MS/MS.
  3. Diagnostics (Basel). 2020 Jul 07. pii: E462. [Epub ahead of print]10(7):
      Bile acids (BA) play a pivotal role in cholesterol metabolism. Their blood concentration has also been proposed as new prognostic and diagnostic indicator of hepatobiliary, intestinal, and cardiovascular disease. Liquid chromatography tandem mass spectrometry (LC-MS/MS) currently represents the gold standard for analysis of BA profile in biological samples. We report here development and validation of a LC-MS/MS technique for simultaneously quantifying 15 BA species in serum samples. We also established a reference range for adult healthy subjects (n = 130) and performed a preliminary evaluation of in vitro and in vivo interference. The method displayed good linearity, with high regression coefficients (>0.99) over a range of 5 ng/mL (lower limit of quantification, LLOQ) and 5000 ng/mL for all analytes tested. The accuracies were between 85-115%. Both intra- and inter-assay imprecision was <10%. The recoveries ranged between 92-110%. Each of the tested BA species (assessed on three concentrations) were stable for 15 days at room temperature, 4 °C, and -20 °C. The in vitro study did not reveal any interference from triglycerides, bilirubin, or cell-free hemoglobin. The in vivo interference study showed that pools obtained from hyper-cholesterolemic patients and hyper-bilirubinemic patients due to post-hepatic jaundice for benign cholestasis, cholangiocarcinoma and pancreatic head tumors had clearly distinct patterns of BA concentrations compared with a pool obtained from samples of healthy subjects. In conclusion, this study proposes a new suitable candidate method for identification and quantitation of BA in biological samples and provides new insight into a number of variables that should be taken into account when investigating pathophysiological changes of BA in human diseases.
    Keywords:  LC–MS/MS; bile acids; interference
  4. Electrophoresis. 2020 Jul 09.
      Bile acids are a group of compounds essential for lipid digestion and absorption with a steroid skeleton and a carboxylate side chain usually conjugated to glycine or taurine. Bile acids are regulatory molecules for a number of metabolic processes and can be used as biomarkers of various disorders. Since the middle of the twentieth century, the detection of bile acids has evolved from simple qualitative analysis to accurate quantification in complicated mixtures. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. This article overviews the literature from the last two decades (2000-2020) and focuses on bile acid analysis in various human biological samples. The methods for sample preparation, including the sample treatment of conventional (blood plasma, blood serum, and urine) and unconventional samples (bile, saliva, duodenal/gastric juice, feces, etc.) are shortly discussed. Eventually, the focus is on novel analytical approaches and methods for each particular biological sample, providing an overview of the microcolumn separation techniques, such as high-performance liquid chromatography, gas chromatography and capillary electrophoresis used in their analysis. This is followed by a discussion on selected clinical applications. This article is protected by copyright. All rights reserved.
    Keywords:  Bile acids; Clinical analysis; GC-MS; HPLC-MS; Microcolumn separation
  5. Anal Chem. 2020 Jul 07.
      Among all the human biological fluids used for disease biomarker discovery or clinical chemistry, urine stands out. It can be col-lected easily and non-invasively, it is readily available in large volumes, it is typically free from protein contamination and it is chemically complex - reflecting a wide range of physiological states and functions. However, the comprehensive metabolomic analysis of urine has been somewhat less studied compared to blood. Indeed, most published metabolomic assays are specifically optimized for serum or plasma. In an effort to improve this situation we have developed a comprehensive, quantitative MS-based assay for urine analysis. The assay robustly detects and quantifies 142 urinary metabolites including 28 amino acids and deriva-tives, 17 organic acids, 22 biogenic amines and derivatives, 40 acylcarnitines, 34 lipids and glucose/hexose, among which 67 me-tabolites are absolutely quantified and 75 metabolites are semi-quantified. All the analysis methods in this assay are based on liq-uid chromatography-tandem mass spectrometry (LC-MS/MS) using both positive and negative-mode multiple reaction monitoring (MRM). The recovery rates of spiked urine samples at three different concentration levels, i.e., low, medium and high, are in the range of 80% to 120% with satisfactory precision values of less than 20%. This targeted metabolomic assay has been successfully applied to the analysis of large numbers of human urine samples, with results closely matching those reported in the literature as well as those obtained from orthogonal analysis via NMR spectroscopy. Moreover, the assay was specifically developed in a 96-well plate format, which enables automated, high-throughput sample analysis. The assay has already been used to analyze more than 1800 urine samples in our laboratory since early 2019.
  6. J Pharm Biomed Anal. 2020 Jun 20. pii: S0731-7085(20)31312-1. [Epub ahead of print]189 113426
      Oxylipins play an important role in cell signaling and they act as auto- and paracrine factors. There are numerous reports on the analysis of oxylipins in biofluids, especially in plasma. Only a limited number of studies addressed the analysis of oxylipins in platelets using modern, sensitive LCMS methods, even though these compounds have a huge impact on platelet functions and thrombo-inflammation. In this work, a new method based on superficially porous particle (2.7 μm) capillary column (0.5 mm ID) and micro-liquid chromatography coupled to tandem mass spectrometry (μUHPLC-ESI-QqQ-MS/MS) method has been developed, optimized and validated. It has finally been successfully applied for human plasma and platelet analysis. The method allows the precise and accurate simultaneous quantification of 42 oxylipins with 13 deuterated internal standards. Solid phase extraction with Bond Elut Certify II provides good extraction recoveries (on average around 75 %). The μUHPLC-MS/MS method is selective, sensitive (LOQs between 30 and 150 pg/mL) and shows good linearity. Limits of detections for most of the compounds are between 2 and 250 fmol on column. Twenty-three oxylipins have been detected in plasma and 19 in non-activated (resting) platelets (all samples were from healthy donors). The μUHPLC-MS/MS method uses very low volume of mobile phase (less than 250 μL of organic solvents in mobile phase per analysis), and therefore is considered environmentally friendly. It also turned out to be robust enough for routine analysis.
    Keywords:  Bioanalysis; Capillary HPLC-ESI-MS/MS; Lipid mediator; Platelet; Superficially porous particle capillary column; Thromboinflammation
  7. BMC Bioinformatics. 2020 Jul 10. 21(1): 297
      BACKGROUND: Stable isotope tracing has become an invaluable tool for probing the metabolism of biological systems. However, data analysis and visualization from metabolic tracing studies often involve multiple software packages and lack pathway architecture. A deep understanding of the metabolic contexts from such datasets is required for biological interpretation. Currently, there is no single software package that allows researchers to analyze and integrate stable isotope tracing data into annotated or custom-built metabolic networks.RESULTS: We built a standalone web-based software, Escher-Trace, for analyzing tracing data and communicating results. Escher-Trace allows users to upload baseline corrected mass spectrometer (MS) tracing data and correct for natural isotope abundance, generate publication quality graphs of metabolite labeling, and present data in the context of annotated metabolic pathways. Here we provide a detailed walk-through of how to incorporate and visualize 13C metabolic tracing data into the Escher-Trace platform.
    CONCLUSIONS: Escher-Trace is an open-source software for analysis and interpretation of stable isotope tracing data and is available at .
    Keywords:  Escher; Metabolism; Stable isotope tracing; Visualization; Web application
  8. Anal Chem. 2020 Jul 09.
      In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the high complex sample comparing to the analyses without FAIMS. Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.
  9. BMC Bioinformatics. 2020 Jul 08. 21(1): 292
      BACKGROUND: Bioinformaticians collaborating with life scientists need software that allows them to involve their collaborators in the process of data analysis.RESULTS: We have developed a web application that allows researchers to publish and execute data analysis scripts. Within the platform bioinformaticians are able to deploy data analysis workflows (recipes) that their collaborators can execute via point and click interfaces. The results generated by the recipes are viewable via the web interface and consist of a snapshot of all the commands, printed messages and files that have been generated during the recipe run. A demonstration version of our software is available at . Detailed documentation for the software is available at: . The source code for the software is distributed through GitHub at .
    CONCLUSIONS: Our software platform supports collaborative interactions between bioinformaticians and life scientists. The software is presented via a web application that provides a high utility and user-friendly approach for conducting reproducible research. The recipes developed and shared through the web application are generic, with broad applicability and may be downloaded and executed on other computing platforms.
    Keywords:  Data analysis; Reproducibility; Scientific workflows
  10. J Sep Sci. 2020 Jul 06.
      Fast, simple, accurate and reproducible RP-HPLC method with direct UV measurement of memantine hydrochloride in tablets was developed, without any chemical derivatization pretreatment. Three main problems appear during chromatographic analysis of memantine: detection, achieving appropriate column retention and limited choice of mobile phase components, as a result of memantine molecular structure. Amongst more than 35 tested columns, the best retention and peak symmetry yielded two C8 and three C18 columns with different characteristics, at a temperature of 30 °C, mobile phase composed of 1% v/v acetonitrile and 99% v/v of 0.05% - 0.1% phosphoric acid or 2.5 - 5μM phosphate buffer, at flow rate 1 ml/min and injection volume of 5 μl. The retention time of memantine was between 2.6 - 4 minutes. Both mobile phase concepts, showed perfect linearity, precision and accuracy. This is the first successful and reproducible direct RP-HPLC-UV quantification method for memantine. This article is protected by copyright. All rights reserved.
    Keywords:  Analytical techniques; Liquid chromatography; Quantification; Reversed Phase
  11. Trends Analyt Chem. 2019 Nov;pii: 115330. [Epub ahead of print]120
      Shotgun lipidomics is one of the most powerful tools in analysis of cellular lipidomes in lipidomics, which directly analyzes lipids from lipid extracts of diverse biological samples with high accuracy/precision. However, despite its great advances in high throughput analysis of cellular lipidomes, low coverage of poorly ionized lipids, especially those species in very low abundance, and some types of isomers within complex lipid extracts by shotgun lipidomics remains a huge challenge. In the past few years, many strategies have been developed to enhance shotgun lipidomics for comprehensive analysis of lipid species. Chemical derivatization represents one of the most attractive and effective strategies, already receiving considerable attention. This review focuses on novel advanced derivatization strategies for enhancing shotgun lipidomics. It is anticipated that with the development of enhanced strategies, shotgun lipidomics can make greater contributions to biological and biomedical research.
    Keywords:  Bis(monoacylglycero) phosphate; chemical derivatization; multi-dimensional mass spectrometry; plasmalogen; polyphosphoinositides; shotgun lipidomics
  12. MethodsX. 2020 ;7 100951
      Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a host of microbiologically-relevant matrices. Here, we provide a detailed bioanalytical protocol that contains several modifications of a method previously described by Wegner et al. [1]. Briefly, this modified method exhibits the following advantages for the measurement of cholic acid (CA), taurocholic acid (TCA), and deoxycholic acid (DCA) in microbiome-relevant sample matrices: i) fecal sample processing has been streamlined by the elimination of lyophilization and manual homogenization steps; ii) the Sciex 6500 QTRAP hybrid triple-quadrupole/linear ion trap mass spectrometer has sufficient sensitivity to perform the measurement of bile acids/salts in negative ion mode - ammonium adducts of bile acids/salts are not required for detection; and, iii) assay throughput has been boosted by more than 5-fold by shortening the chromatographic duty cycle of a single sample injection from 45 min to 8.4 min. Recently, the method was used to perform 508 sequential injections (72 calibration standards, 52 blank-internal standard sample, and 368 MiniBioReactor Array (MBRA)-derived samples) from four separate batches over a 4-day time period.
    Keywords:  Bioanalytical chemistry; High-performance liquid chromatography; High-throughput sample analysis; LC-MS/MS; Microbiology; Microbiome
  13. Phytochem Anal. 2020 Jul 07.
      INTRODUCTION: Pentacyclic triterpenoids (PCTs) are secondary plant metabolites. They are of exceptional interest as biologically active substances and raw materials for a wide range of medications. Thus, the development of a methodology for rapid screening of PCTs in plant biomass is an important task.OBJECTIVE: The goal of this work was to develop an approach for simultaneous screening and semi-quantitative determination of PCTs in plant tissues by liquid chromatography-tandem mass spectrometry with a precursor ion scan (PrecIS).
    MATERIALS AND METHODS: Pressurised liquid extraction (PLE) with methanol was used for the isolation of PCTs from plant biomass. Screening and semi-quantitative determination of PCTs in the obtained extracts were carried out by reversed phase high-performance liquid chromatography-tandem mass spectrometry in a PrecIS mode.
    RESULTS: The product ion at m/z 95 with collision energy of 40 V was used as a diagnostic ion to identify PCTs by the PrecIS mode. In plant materials, 26 PCTs and their derivatives, such as PCTs esters and glycosides, were detected and identified. Calculation of the relative response factor for nine available PCTs showed that using a betulin calibration curve allows us to estimate the semi-quantitative content of PCTs and their derivatives in plant PLE extracts.
    CONCLUSION: The developed approach can be applied for simultaneous untargeted screening and semi-quantitative determination of PCTs and their derivatives in various plants at sub-parts per million levels.
    Keywords:  liquid chromatography-mass spectrometry; pentacyclic triterpenoids; plant feedstock; precursor ion scan; screening
  14. J Mass Spectrom. 2020 May 31. e4583
      The performance of a compact high-pressure electrospray ionization (HP-ESI) source that can be readily used for commercial atmospheric pressure ionization (API) mass spectrometers is reported. The ion source employs a converging-diverging outlet nozzle, and ions/droplets generated inside the high-pressure compartment are carried by the high-velocity air jet toward the mass spectrometry (MS) ion inlet placed under the atmospheric pressure. With the use of a shielding electrode, the HP-ESI can also be operated with its emitter held at ground potential. This feature prevents the flow of current from the emitter to other electrically grounded components and facilitates the connection of ion source to liquid chromatography (LC) columns or capillary electrophoresis. Sensitive detection of proteins from highly conductive aqueous solutions such as 0.1% trifluoroacetic acid (TFA) solution and the prevention of electrochemical artifacts by the grounded emitter operation are demonstrated.
    Keywords:  converging-diverging nozzle; grounded ESI emitter; high-pressure electrospray; highly conductive aqueous solutions; native MS; protein oxidation
  15. Molecules. 2020 Jul 03. pii: E3047. [Epub ahead of print]25(13):
      In recent decades, mass spectrometry techniques, particularly when combined with separation methods such as high-performance liquid chromatography, have become increasingly important in pharmaceutical, bio-analytical, environmental, and food science applications because they afford high selectivity and sensitivity. However, mass spectrometry has limitations due to the matrix effects (ME), which can be particularly marked in complex mixes, when the analyte co-elutes together with other molecules, altering analysis results quantitatively. This may be detrimental during method validation, negatively affecting reproducibility, linearity, selectivity, accuracy, and sensitivity. Starting from literature and own experience, this review intends to provide a simple guideline for selecting the best operative conditions to overcome matrix effects in LC-MS techniques, to obtain the best result in the shortest time. The proposed methodology can be of benefit in different sectors, such as pharmaceutical, bio-analytical, environmental, and food sciences. Depending on the required sensitivity, analysts may minimize or compensate for ME. When sensitivity is crucial, analysis must try to minimize ME by adjusting MS parameters, chromatographic conditions, or optimizing clean-up. On the contrary, to compensate for ME analysts should have recourse to calibration approaches depending on the availability of blank matrix. When blank matrices are available, calibration can occur through isotope labeled internal standards and matrix matched calibration standards; conversely, when blank matrices are not available, calibration can be performed through isotope labeled internal standards, background subtraction, or surrogate matrices. In any case, an adjusting of MS parameters, chromatographic conditions, or a clean-up are necessary.
    Keywords:  HPLC-MS; matrix effect; method validation
  16. Comb Chem High Throughput Screen. 2020 Jul 09.
      BACKGROUND: Terbinafine is an allylamine antifungal which is effective against many fungi, dermatophytes and moulds. Analytical methods are required for the determination of terbinafine in biological fluids to perform therapeutic drug monitoring and pharmacokinetic studies.OBJECTIVE: The aim of this study was to develop and validate a novel and fast method combining dilute and shoot approach and high-performance liquid chromatography coupled with photodiode array detection for the determination of terbinafine in human urine.
    METHODS: Chromatographic parameters including mobile phase composition, pH, flow rate and injection volume was assessed and optimized. The separation of terbinafine and naproxen (internal standard) was achieved within 3 min using a C18 core-shell column (Raptor ARC-18, 100 x 4.6 mm, 2.7 µm) under isocratic conditions. Samples were eluted from the column at the flow rate of 1.4 mL/min using a mobile phase containing 0.2% triethylamine in water (pH 3.4 with formic acid): acetonitrile (45:55, v/v).
    RESULTS: Presented technique was linear in the range of 25-2000 ng/mL. Intra- and inter-day reproducibility at four quality control levels (25, 200, 750 and 1500 ng/mL) was less than 7%, with relative errors ranging from -5.40% to 5.91%. Limit of detection was 12.60 ng/mL. Developed method has three main advantages compared to existing methods: simplicity and greenness of sample preparation, use of core-shell column and short analysis time.
    CONCLUSION: The results of this study indicate that the combination of dilute and shoot approach and core-shell column can be regarded as an advantageous application for the fast determination of terbinafine in urine.
    Keywords:  Core-shell columns; Dilute and shoot approach; Fast analysis; High performance liquid chromatography; Terbinafine; Urine.