bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒10‒11
seventeen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Environ Sci Pollut Res Int. 2020 Oct 09.
      A simplified method is described for reducing the analysis time of nitrofurans (NFs) and nitrofuran metabolites (NFMs) in the aquatic animals. Most existing HPLC-MS/MS methods are intended only for NFMs and are based on their fast metabolic transformations. We optimized a method for simultaneously detecting major NFs and their metabolites, including nitrovin (NV) that imply use of an optimized buffer solution. The novel method was validated by six different aquatic animal matrices (loach, catfish, shrimp, lobster, scallop, and eel) spiked with the analytes at 0.5, 1.0, and 2.0 μg kg-1. Recovery rates and %RSDs (relative standard deviations) of 82-97% and 1-8% were observed for NFMs, respectively, with values of 70-96% and 1-8% obtained for furazolidone, furaltadone, nitrofurazone, nitrofurantoin, and NV, respectively. Linearity was observed in the 0.1-20 μg L-1 range, with correlation coefficients greater than 0.99 recorded for all compounds. The developed method is sensitive, accurate, easier to use, and faster than previous methods when applied to real samples. To the best of our knowledge, this is the first method that can simultaneously determine NFs and their metabolites, as well as NV, using a single-step extraction process.
    Keywords:  HPLC-MS/MS; Nitrofuran, nitrofuran metabolites; Nitrovin; Residue analysis; Veterinary drug
  2. Forensic Sci Int. 2020 Sep 13. pii: S0379-0738(20)30368-6. [Epub ahead of print]317 110506
      A study of impaired driving rates in the province of Québec is currently planned following the legalization of recreational cannabis in Canada. Oral fluid (OF) samples are to be collected with a Quantisal® device and sent to the laboratory for analysis. In order to prepare for this project, a qualitative decision point analysis method monitoring for the presence of 97 drugs and metabolites in OF was developed and validated. This high throughput method uses incubation with a precipitation solvent (acetone:acetonitrile 30:70 v:v) to boost drug recovery from the collecting device and improve stability of benzodiazepines (e.g., α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, nitrazepam). The Quantisal® device has polyglycol in its stabilizing buffer, but timed use of the mass spectrometer waste valve proved sufficient to avoid the glycol interferences for nearly all analytes. Interferences from OF matrices and 140 potentially interfering compounds, carryover, ion ratios, stability, recovery, reproducibility, robustness, false positive rate, false negative rate, selectivity, sensitivity and reliability rates were tested in the validation process. Five of the targeted analytes (olanzapine, oxazepam, 7-aminoclonazepam, flunitrazepam and nitrazepam) did not meet the set validation criteria but will be monitored for identification purposes (no comparison to a cut-off level). Blind internal proficiency testing was performed, where six OF samples were tested and analytes were classified as "negative", "likely positive" or "positive" with success. The final validated OF qualitative decision point method covers 92 analytes, and the presence of 5 additional analytes is screened in this high throughput analysis.
    Keywords:  Liquid chromatography tandem mass spectrometry; Oral fluid; Qualitative decision point methods; Quantisal; Roadside survey; Saliva
  3. Ocul Surf. 2020 Oct 03. pii: S1542-0124(20)30148-8. [Epub ahead of print]
      Human tear is a biological fluid rich in lipids that is increasingly collected in clinical and biological research. The repertoire of small lipids or lipid mediators (often termed eicosanoids or oxylipins) found in human tear provides insight into metabolism of fatty acids and physiology of the ocular surface and Meibomian glands. Disturbances in the tear lipid mediators profile also occur during inflammation of the ocular surface that is not directly linked to lipid metabolism. The changes in the levels of pro-inflammatory and pro-resolution lipid mediators in the tear help assess the severity and stage of inflammation in ocular surface tissues. Mass spectrometry, used in the evaluation of tear lipid mediators, is an emerging tool in clinical diagnostics and personalized medicine. Here we describe the reproducibility, accuracy, and precision of quantifying lipid mediators in human tears, with a suggested method for tear collection and sample handling. The ranges of lipid mediators concentrations in tear fluid of healthy and diseased individuals with Meibomian gland dysfunction are reported, as well as the impact of age and disease on individual lipid mediators. We would like to recommend a set of guidelines, which can be further discussed in workshops. This will facilitate harmonization of future tear lipid mediators data across different instrument platforms in various laboratories. We hope that other fields requiring lipid mediators assays will also benefit from such an effort.
    Keywords:  Clinical guidelines; Dry eye; Lipid mediators; Meibomian gland dysfunction; Ocular surface inflammation; Tear
  4. J Pharm Biomed Anal. 2020 Sep 23. pii: S0731-7085(20)31532-6. [Epub ahead of print]192 113646
      The pseudotargeted lipidomics method integrates the advantages of untargeted andtargeted lipidomics methods as a novel emerging approach. In this study, a green andefficient pseudotargeted lipidomics method based on ultra-high performancesupercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) wasdeveloped. The tandem mass spectra of the analytes were obtained by using UHPSFCwith quadrupole-time of flight MS (Q-TOF MS) in MS E mode and the multiplereaction monitoring (MRM) transitions of the lipidome were defined. Then, thecandidate MRM transitions were verified by UHPSFC with triple quadrupole massspectrometry (QqQ MS) in the scheduled MRM mode. In total, 758 potential lipidscorresponding to 509 and 249 MRM transitions were detected within 8 min in positiveand negative modes, respectively. The established pseudotargeted lipidomics methodwas validated to have excellent analytical characteristics. Compared with thepseudotargeted method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), the UHPSFC-MS/MS-basedpseudotargeted method not only reduced the analytical time by half but also improvedthe sensitivity and resolution for most analytes, especially had better separation forlipid isomers. Besides, the UHPSFC-MS/MS-based pseudotargeted method showedhigher sensitivity and better repeatability for most analytes than the UHPSFC-MS/MS-based untargeted method. The established method was finally applied to investigatingthe lipid profiles of the plasma from the depressed rats and 33 differential variableswere screened, which related to three metabolic pathways. The results indicated thatthe UHPSFC-MS/MS-based pseudotargeted method is reliable and efficient and couldbe used in the lipidomics studies.
    Keywords:  Depression; Liquiritin; Pseudotargeted lipidomics method; Ultra-high performance supercritical fluid chromatography-tandem mass spectrometry
  5. Metabolomics. 2020 Oct 07. 16(10): 107
      INTRODUCTION: It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites.OBJECTIVES: Blood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines.
    METHODS: We develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the 'exometabolome' or metabolic footprint).
    RESULTS: Our method measures some 4000-5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other.
    CONCLUSION: Our new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds.
    Keywords:  Cell culture; Human serum; LC-MS/MS; Orbitrap; Transporters; Untargeted metabolomics
  6. BMC Bioinformatics. 2020 Oct 07. 21(1): 444
      BACKGROUND: Metabolomics data analyses rely on the use of bioinformatics tools. Many integrated multi-functional tools have been developed for untargeted metabolomics data processing and have been widely used. More alternative platforms are expected for both basic and advanced users.RESULTS: Integrated mass spectrometry-based untargeted metabolomics data mining (IP4M) software was designed and developed. The IP4M, has 62 functions categorized into 8 modules, covering all the steps of metabolomics data mining, including raw data preprocessing (alignment, peak de-convolution, peak picking, and isotope filtering), peak annotation, peak table preprocessing, basic statistical description, classification and biomarker detection, correlation analysis, cluster and sub-cluster analysis, regression analysis, ROC analysis, pathway and enrichment analysis, and sample size and power analysis. Additionally, a KEGG-derived metabolic reaction database was embedded and a series of ratio variables (product/substrate) can be generated with enlarged information on enzyme activity. A new method, GRaMM, for correlation analysis between metabolome and microbiome data was also provided. IP4M provides both a number of parameters for customized and refined analysis (for expert users), as well as 4 simplified workflows with few key parameters (for beginners who are unfamiliar with computational metabolomics). The performance of IP4M was evaluated and compared with existing computational platforms using 2 data sets derived from standards mixture and 2 data sets derived from serum samples, from GC-MS and LC-MS respectively.
    CONCLUSION: IP4M is powerful, modularized, customizable and easy-to-use. It is a good choice for metabolomics data processing and analysis. Free versions for Windows, MAC OS, and Linux systems are provided.
    Keywords:  Data analysis; Metabolomics; Software; Workflow
  7. Crit Rev Anal Chem. 2020 Oct 07. 1-23
      Metabolomics is a young field of knowledge that arises linked to other omics such as genomics, transcriptomics, and proteomics. This discipline seeks to understand the performance of metabolites, identifying, quantifying them, and thus understanding its mechanism of action. This new branch of omics science shows high potential, due to its noninvasive character and its close relation with phenotype. Several techniques have been developed to study the metabolome of biological samples, fundamentally nuclear magnetic resonance (NMR), mass spectrometry (MS) and vibrational spectrometry (VS) or a combination of several techniques. These techniques are focused to separate, detect, characterize, and quantify metabolites, as well as elucidate their structures and their function on the metabolic pathways they are involved. However, due to the complexity of the metabolome, in most cases it is necessary to apply several of these techniques to understand completely the whole scenery. This review is aimed to offer a summary of the current knowledge of these analytical techniques for metabolomics and their application to different fields as environmental, food or health sciences. Each technique shows different advantages and drawbacks depending on their technical characteristics and limitations, some factors, such as the aim of the study or the nature of the biological sample will condition the choice. Regarding their applications, NMR has been employed specially to identify new compounds and elucidate structures. The use of MS has gained popularity because of its versatility, easiness to be coupled to separation techniques and its high sensitivity. Whereas VS is widely employed for in situ studies, due to its nondestructive character. Metabolomics applications in different science fields are growing each year, due to advances in analytical techniques and combination with other omics that allow to increase the comprehension of metabolic processes. Further development of analytical tools is necessary to continue exploiting all the possibilities of metabolomics. Highlights Metabolomics seeks to understand the performance of metabolites and its mechanism of action Different metabolomics techniques have been developed and improved in the last years Metabolomics applications cover clinical, pharmaceuticals and food and environmental sciences This review is aimed to offer a summary of the current knowledge of these analytical techniques.
    Keywords:  Metabolomics; applications; mass spectrometry; metabolite; nuclear magnetic resonance; vibrational spectroscopy
  8. J Anal Toxicol. 2020 Oct 07. pii: bkaa148. [Epub ahead of print]
      Bone marrow is the tissue contained inside the bones and can be considered one of the potential alternative tissues in forensic toxicology. This matrix could be particularly useful in those cases where the routine sample is not available due to an advanced state of decomposition or skeletonization of the corpse. Aim of this study was develop, validate and apply an analytical method of extraction and analysis of different antidepressants and antipsychotics, commonly used in therapy, from spiked pig ribs. Pig ribs, each of 5 g and 5 cm long, were spiked at three concentration levels (100, 200 and 500 ng/g). For each concentration, ten pig ribs were prepared. The method involves the leaching by ethanol at different pHs of nine drugs from the inside of ribs, in particular from the bone marrow, without the fragmentation of bone tissue. Following a liquid-liquid purification and extraction, analysis was performed by gas chromatography coupled with mass spectrometry in SIM mode. For method validation was assessed linearity, sensitivity, precision and accuracy, matrix interferences and, finally, carryover. Analytical method performance was acceptable respect to acceptance criteria for validation. No matrix interferences were detected; for this reason, it is possible to affirm that this method has a good selectivity. Moreover, the method was not affected by carryover. Considering that the study conducted on pig ribs has given encouraging results, it can be assumed that this method can be used in forensic toxicological protocols (human autopsy cases) as alternative to classic procedures.
    Keywords:  Antidepressant; Antipsychotics; Autopsy; Bone marrow; Drugs; Forensic toxicology; Pig ribs
  9. J Lipid Res. 2020 Oct 09. pii: jlr.S120001025. [Epub ahead of print]
      A comprehensive and standardized system to report lipid structures analyzed by mass spectrometry is essential for the communication and storage of lipidomics data. Herein, an update on both the LIPID MAPS classification system and shorthand notation of lipid structures is presented for lipid categories Fatty Acyls (FA), Glycerolipids (GL), Glycerophospholipids (GP), Sphingolipids (SP), and Sterols (ST). With its major changes, i.e. annotation of ring double bond equivalents and number of oxygens, the updated shorthand notation facilitates reporting of newly delineated oxygenated lipid species as well. For standardized reporting in lipidomics, the hierarchical architecture of shorthand notation reflects the diverse structural resolution powers provided by mass spectrometric assays. Moreover, shorthand notation is expanded beyond mammalian phyla to lipids from plant and yeast phyla. Finally, annotation of atoms is included for the use of stable isotope-labelled compounds in metabolic labelling experiments or as internal standards.This update on lipid classification, nomenclature and shorthand annotation for lipid mass spectra is considered a standard for lipid data presentation.
    Keywords:  Glycerolipids; Glycerophospholipids; Lipidomics; Mass spectrometry; Sphingolipids; Sterols
  10. Metabolites. 2020 Oct 02. pii: E393. [Epub ahead of print]10(10):
      The field of breath analysis lacks a fully automated analysis platform that enforces machine learning good practice and enables clinicians and clinical researchers to rapidly and reproducibly discover metabolite patterns in diseases. We present BALSAM-a comprehensive web-platform to simplify and automate this process, offering features for preprocessing, peak detection, feature extraction, visualization and pattern discovery. Our main focus is on data from multi-capillary-column ion-mobility-spectrometry. While not limited to breath data, BALSAM was developed to increase consistency and robustness in the data analysis process of breath samples, aiming to expand the array of low cost molecular diagnostics in clinics. Our platform is freely available as a web-service and in form of a publicly available docker container.
    Keywords:  biomarker; breath analysis; data-mining; machine learning; metabolite discovery; metabolomics; web-platform
  11. Bioinformatics. 2020 Oct 07. pii: btaa856. [Epub ahead of print]
      We present LipidFinder 2.0, incorporating four new modules that apply artefact filters, remove lipid and contaminant stacks, in-source fragments and salt clusters, and a new isotope deletion method which is significantly more sensitive than available open-access alternatives. We also incorporate a novel false discovery rate (FDR) method, utilizing a target-decoy strategy, which allows users to assess data quality. A renewed lipid profiling method is introduced which searches three different databases from LIPID MAPS and returns bulk lipid structures only, and a lipid category scatter plot with color blind friendly pallet. An API interface with XCMS Online is made available on LipidFinder's online version. We show using real data that LipidFinder 2.0 provides a significant improvement over non-lipid metabolite filtering and lipid profiling, compared to available tools.AVAILABILITY: LipidFinder 2.0 is freely available at and
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  12. Sci Rep. 2020 Oct 05. 10(1): 16512
      Desorption electrospray ionisation mass spectrometry (DESI-MS) can image hundreds of molecules in a 2D tissue section, making it an ideal tool for mapping tumour heterogeneity. Tumour lipid metabolism has gained increasing attention over the past decade; and here, lipid heterogeneity has been visualised in a glioblastoma xenograft tumour using 3D DESI-MS imaging. The use of an automatic slide loader automates 3D imaging for high sample-throughput. Glioblastomas are highly aggressive primary brain tumours, which display heterogeneous characteristics and are resistant to chemotherapy and radiotherapy. It is therefore important to understand biochemical contributions to their heterogeneity, which may be contributing to treatment resistance. Adjacent sections to those used for DESI-MS imaging were used for H&E staining and immunofluorescence to identify different histological regions, and areas of hypoxia. Comparing DESI-MS imaging with biological staining allowed association of different lipid species with hypoxic and viable tissue within the tumour, and hence mapping of molecularly different tumour regions in 3D space. This work highlights that lipids are playing an important role in the heterogeneity of this xenograft tumour model, and DESI-MS imaging can be used for lipid 3D imaging in an automated fashion to reveal heterogeneity, which is not apparent in H&E stains alone.
  13. Magn Reson Chem. 2020 Oct 08.
      NMR-based metabolomics has witnessed rapid advancements in recent years with the continuous development of new methods to enhance the sensitivity, resolution and speed of data acquisition. Some of the approaches were earlier used for peptide and protein resonance assignments and have now been adapted to metabolomics. At the same time, new NMR methods involving novel data acquisition techniques, suited particularly for high-throughput analysis in metabolomics, have been developed. In this review, we focus on the different sampling strategies or data acquisition methods that have been developed in our laboratory and other groups to acquire NMR spectra rapidly with high sensitivity and resolution for metabolomics. In particular, we focus on the use of multiple receivers, phase modulation NMR spectroscopy and fast pulsing methods for identification and assignments of metabolites.
    Keywords:  Fast pulsing; Metabolomics; Multiple receiver NMR; Phase modulation
  14. Mol Imaging Biol. 2020 Oct 06.
      Mass spectrometry imaging (MSI) enables the visualization of molecular distributions on complex surfaces. It has been extensively used in the field of biomedical research to investigate healthy and diseased tissues. Most of the MSI studies are conducted in a 2D fashion where only a single slice of the full sample volume is investigated. However, biological processes occur within a tissue volume and would ideally be investigated as a whole to gain a more comprehensive understanding of the spatial and molecular complexity of biological samples such as tissues and cells. Mass spectrometry imaging has therefore been expanded to the 3D realm whereby molecular distributions within a 3D sample can be visualized. The benefit of investigating volumetric data has led to a quick rise in the application of single-sample 3D-MSI investigations. Several experimental and data analysis aspects need to be considered to perform successful 3D-MSI studies. In this review, we discuss these aspects as well as ongoing developments that enable 3D-MSI to be routinely applied to multi-sample studies.
    Keywords:  3D imaging; Data analysis; Experimental set-up; Mass spectrometry imaging
  15. J Chromatogr A. 2020 Sep 28. pii: S0021-9673(20)30860-8. [Epub ahead of print]1631 461586
      The presence of pharmaceuticals and personal care products (PPCPs) in aquatic systems has raised concern about their potential adverse effects on aquatic organisms. Considering the fact that the physiological/biological effects of PPCPs are triggered when their concentrations in the organism exceeds the respective threshold values, it is important to understand the bioconcentration and toxicokinetics of PPCPs in aquatic organisms. In the present study, we developed a convenient analytical method for the determination of 65 pharmaceuticals and 7 personal care products (log Kow = 0.14-6.04) in plasma and whole-body tissues of fish. The analytical method consists of ultrasound-assisted extraction in methanol/acetonitrile (1:1, v/v,) acidified with acetic acid-ammonium acetate buffer (pH 4), cleanup on a HybridSPE®-Phospholipid cartridge (zirconia-coated silica cartridge), and quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Acceptable accuracy (internal standard-corrected recovery: 70%-120%) and intra- and inter-day precision (coefficient of variation: <15%) were obtained for both plasma and whole-body tissue samples. In addition, low method detection limits were achieved for both plasma (0.0077 to 0.93 ng mL-1) and whole-body tissue (0.022 to 4.3 ng g - 1 wet weight), although the developed method is simple and fast - a batch of 24 samples can be prepared within 6 h, excluding the time for measurement with LC-MS/MS. The developed method was successfully applied to the analysis of PPCPs in plasma and whole-body tissue samples of fish collected in a treated wastewater-dominated stream, for a comprehensive evaluation of their bioconcentration properties. The analytical method developed in the present study is sufficiently accurate, sensitive, and rapid, and thus highly useful for the comprehensive evaluation of PPCP residues in fish and would aid in future exposome and risk assessment.
    Keywords:  Fish; HybridSPE®-Phospholipid cartridge; Personal care products; Pharmaceuticals; Rapid analysis
  16. J Pharm Biomed Anal. 2020 Aug 28. pii: S0731-7085(20)31476-X. [Epub ahead of print]191 113590
      Quantification of pharmaceutical compounds in skin tissue is challenging because of low expected concentrations, small typical sample volumes, and the hard nature of the skin structure itself. This review provides a comprehensive overview of sample collection, sample homogenization and analyte extraction methods that have been used to quantify pharmaceutical compounds in skin tissue, obtained from animals and humans, using liquid chromatography-mass spectrometry. For each step in the process of sample collection to sample extraction, methods are compared to discuss challenges and provide practical guidance. Furthermore, liquid chromatographic-mass spectrometry considerations regarding the quality and complexity of skin tissue sample measurements are discussed, with emphasis on analyte recovery and matrix effects. Given that the true recovery of analytes from skin tissue is difficult to assess, the extent of homogenization plays a crucial role in the accuracy of quantification. Chemical or enzymatic solubilization of skin tissue samples would therefore be preferable as homogenization method.
    Keywords:  Bioanalysis; Dermal; Liquid chromatography tandem mass spectrometry; Quantification; Sample preparation; Tissue homogenization
  17. BMC Bioinformatics. 2020 Oct 09. 21(1): 448
      BACKGROUND: Multimodal imaging that combines mass spectrometry imaging (MSI) with Raman imaging is a rapidly developing multidisciplinary analytical method used by a growing number of research groups. Computational tools that can visualize and aid the analysis of datasets by both techniques are in demand.RESULTS: Raman2imzML was developed as an open-source converter that transforms Raman imaging data into imzML, a standardized common data format created and adopted by the mass spectrometry community. We successfully converted Raman datasets to imzML and visualized Raman images using open-source software designed for MSI applications.
    CONCLUSION: Raman2imzML enables both MSI and Raman images to be visualized using the same file format and the same software for a straightforward exploratory imaging analysis.
    Keywords:  Converter; Mass spectrometry imaging; R; Raman imaging; imzML