bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒09‒13
eleven papers selected by
Sofia Costa
Cold Spring Harbor Laboratory


  1. Metabolomics. 2020 Sep 11. 16(9): 99
    Wolthuis JC, Magnusdottir S, Pras-Raves M, Moshiri M, Jans JJM, Burgering B, van Mil S, de Ridder J.
      Direct infusion untargeted mass spectrometry-based metabolomics allows for rapid insight into a sample's metabolic activity. However, analysis is often complicated by the large array of detected m/z values and the difficulty to prioritize important m/z and simultaneously annotate their putative identities. To address this challenge, we developed MetaboShiny, a novel R/RShiny-based metabolomics package featuring data analysis, database- and formula-prediction-based annotation and visualization. To demonstrate this, we reproduce and further explore a MetaboLights metabolomics bioinformatics study on lung cancer patient urine samples. MetaboShiny enables rapid and rigorous analysis and interpretation of direct infusion untargeted mass spectrometry-based metabolomics data.
    Keywords:  Annotation; Direct infusion; Machine learning; Mass spectrometry; Metabolomics; R; Statistics
    DOI:  https://doi.org/10.1007/s11306-020-01717-8
  2. Zhongguo Zhong Yao Za Zhi. 2020 Aug;45(16): 3952-3960
    Lou TY, Ma BB, Liang YY, Wang CX, Liu JH, Li RJ, Yu SY, Wang ZB.
      A method of ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field Obitrap high-resolution mass spectrometry(UHPLC-Q-Exactive MS) was established to comprehensively identify the metabolites of carnosic acid in rats. After oral gavage of carnosic acid CMC-Na suspension in rats, urine, plasma and feces samples were collected and pretreated by solid phase extraction(SPE). Acquity UPLC BEH C_(18 )column(2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid solution(A)-acetonitrile(B) as the mobile phase for the gradient elution. Biological samples were analyzed by quadrupole/electrostatic field Obitrap high-resolution mass spectrometry in positive and negative ion mode. Based on the accurate molecular mass, fragment ion information, and related literature reports, a total of 28 compounds(including carnosic acid) were finally identified in rat samples. As a result, the main metabolic pathways of carnosic acid in rats are oxidation, hydroxylation, methylation, glucuronide conjugation, sulfate conjugation, S-cysteine conjugation, glutathione conjugation, demethylation, decarbonylation and their composite reactions. The study showed that the metabolism of carnosic acid in rats could be efficiently and comprehensively clarified by using UHPLC-Q-Exactive MS, providing a reference for clarifying the material basis and metabolic mechanism of carnosic acid.
    Keywords:  UHPLC-Q-Exactive MS; carnosic acid; metabolic pathways; metabolites
    DOI:  https://doi.org/10.19540/j.cnki.cjcmm.20200519.201
  3. Clin Lab. 2020 Sep 01. 66(9):
    Enko D, Zelzer S, Baranyi A, Herrmann M, Meinitzer A.
      BACKGROUND: As trimethylamine-N-oxide (TMAO) is considered to be associated with various diseases, rapid determination of serum TMAO concentration is of clinical interest. This study is aimed at evaluating the analytical performance of a simple isocratic liquid chromatography-tandem mass-spectrometry (LC-MS/MS) method for TMAO quantification.METHODS: TMAO measurements were performed on a tandem mass spectrometer, SCIEX QTRAP 4500 (Applied Biosystems, Framingham, MA, USA), coupled with an Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA). The separation was performed on a Hypercarb Porus Grahitic Carbon (PGC) column (ThermoFisher Scientific, Waltham, MA, USA) by isocratic elution mode. Linearity, precision, recovery, and pre-analytical requirements of the TMAO LC-MS/MS method were evaluated. The imprecision acceptance criteria were defined 15%. We investigated sample stability at room temperature (RT) and assessed the serum TMAO concentrations of 188 healthy adults.
    RESULTS: The TMAO LC-MS/MS method was linear over the concentration range of 0.5 - 80 μmol/L. Intra- and inter-day precision ranged between 2.24 - 3.37% and 6.95 - 9.97%, recovery between 106 - 114%, respectively. At RT, serum samples were stable for 8 days. The median serum TMAO concentration of 188 healthy adults was 2.27 μmol/L (2.5th and 97.5th percentile: 0.75 - 10.46 μmol/L), respectively.
    CONCLUSIONS: The isocratic TMAO LC-MS/MS shows a broad analytical range and meets the imprecision acceptance criteria of 15%. This method is a robust and reliable diagnostic tool for the assessment of the human TMAO status. Serum samples are stable at RT for at least 8 days.
    DOI:  https://doi.org/10.7754/Clin.Lab.2020.200122
  4. Anal Chim Acta. 2020 Sep 08. pii: S0003-2670(20)30748-0. [Epub ahead of print]1129 143-149
    Wang C, Wang J, Qin C, Han X.
      Lipid species possess very different structures, leading to their very diversified cellular functions in biological systems. Lipidomics represents a powerful technology for deep analysis of hundreds to thousands of intact lipid molecular species. In the current study, a cluster of unknown ion peaks was displayed when we profiled cerebroside species in rat spinal cord samples by neutral loss scan of 162 Da in the positive ion mode using a multi-dimensional mass spectrometry-based shotgun lipidomics strategy. In order to identify the structural identities of these unknown ion peaks, MS2 and MS3 analyses of these ions were performed by high mass resolution mass spectrometry. Extensive lines of evidence allowed us to identify that these unknown ion peaks were monohexosyl alkyl-acyl glycerol (HAAG) species, including their sn-positional isomers and alkyl-acyl compositional isomers. We also applied the developed method to identify and quantify HAAG species present in a variety of mouse nerve tissues. We believe that the first kind of lipidomics study on HAAG species present in mammalian nerve tissue samples provided the foundation for future biological research in this unknown area.
    Keywords:  Glycolipids; Lipids; Mass spectrometry; Monohexosyl alkyl-acyl glycerol; Neurodegeneration; Shotgun lipidomics
    DOI:  https://doi.org/10.1016/j.aca.2020.07.016
  5. Anal Chim Acta. 2020 Sep 08. pii: S0003-2670(20)30737-6. [Epub ahead of print]1129 31-39
    Randolph CE, Marshall DL, Blanksby SJ, McLuckey SA.
      Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous bioactive lipids with anti-diabetic and anti-inflammatory effects. Identification of FAHFAs is challenging due to both the relatively low abundance of these metabolites in most biological samples and the significant structural diversity arising from the co-occurrence of numerous regioisomers. Ultimately, development of sensitive analytical techniques that enable rapid and unambiguous identification of FAHFAs is integral to understanding their diverse physiological functions in health and disease. While a battery of mass spectrometry (MS) based methods for complex lipid analysis has been developed, FAHFA identification presents specific challenges to conventional approaches. Notably, while the MS2 product ion spectra of [FAHFA - H]¯ anions afford the assignment of fatty acid (FA) and hydroxy fatty acid (HFA) constituents, FAHFA regioisomers are usually indistinguishable by this approach. Here, we report the development of a novel MS-based technique employing charge inversion ion/ion reactions with tris-phenanthroline magnesium complex dications, Mg(Phen)32+, to selectively and efficiently derivatize [FAHFA - H]¯ anions in the gas phase, yielding fixed-charge cations. Subsequent activation of [FAHFA - H + MgPhen2]+ cations yield product ions that facilitate the assignment of FA and HFA constituents, pinpoints unsaturation sites within the FA moiety, and elucidates ester linkage regiochemistry. Collectively, the presented approach represents a rapid, entirely gas-phase method for near-complete FAHFA structural elucidation and confident isomer discrimination without the requirement for authentic FAHFA standards.
    Keywords:  FAHFAs; Gas-phase charge inversion; Shotgun lipidomics; Tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.aca.2020.07.005
  6. Anal Bioanal Chem. 2020 Sep 10.
    Mulet CT, Tarifa A, DeCaprio AP.
      The class of novel psychoactive substances known as synthetic cannabinoids (SC) includes illicit compounds that are sprayed on plant material and smoked or sold as liquids to be vaporized in e-cigarettes. In toxicological analysis of SC, fast analytical methods are needed for the detection and confirmation of parent drugs and metabolites at very low levels. While various analytical methods have been developed for SC in blood and urine, few are available for alternative matrices such as oral fluid (OF). There are numerous advantages to using OF as a sample matrix for SC analysis, including non-invasive collection, lesser risk of adulteration, and presence of both parent drug and metabolites. Here we report a validated online solid-phase extraction (online SPE) method coupled to LC-QqQ-MS for rapid confirmation and quantitation of 72 structurally diverse SC parent drugs and metabolites in OF with 2.5 min of preconcentration time and a total elution time of < 10 min. The use of online SPE for sample pretreatment facilitates rapid and consistent processing and greatly increases sample throughput. The method was fully validated according to relevant guidelines (ANSI/ASB Standard 036). Bias and precision values were within ± 20% for all compounds in human OF matrix. Method detection and quantitation limits ranged from 0.4 to 3.8 ng/mL and from 1.1 to 11.6 ng/mL, respectively. Recovery, matrix effects, process efficiency, carryover, and stability were also within acceptable limits for the majority of compounds. Successful application of the method was demonstrated using blank human OF fortified with SC in addition to a set of authentic OF specimens previously tested by another laboratory. Graphical abstract.
    Keywords:  LC-QqQ-MS; Metabolites; Method validation; Online SPE; Oral fluid; Synthetic cannabinoids
    DOI:  https://doi.org/10.1007/s00216-020-02926-9
  7. Molecules. 2020 Sep 03. pii: E4026. [Epub ahead of print]25(17):
    Tuzimski T, Petruczynik A.
      Therapeutic drug monitoring (TDM) is a tool used to integrate pharmacokinetic and pharmacodynamics knowledge to optimize and personalize various drug therapies. The optimization of drug dosing may improve treatment outcomes, reduce toxicity, and reduce the risk of developing drug resistance. To adequately implement TDM, accurate and precise analytical procedures are required. In clinical practice, blood is the most commonly used matrix for TDM; however, less invasive samples, such as dried blood spots or non-invasive saliva samples, are increasingly being used. The choice of sample preparation method, type of column packing, mobile phase composition, and detection method is important to ensure accurate drug measurement and to avoid interference from matrix effects and drug metabolites. Most of the reported procedures used liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques due to its high selectivity and sensitivity. High-performance chromatography with ultraviolet detection (HPLC-UV) methods are also used when a simpler and more cost-effective methodology is desired for clinical monitoring. The application of high-performance chromatography with fluorescence detection (HPLC-FLD) with and without derivatization processes and high-performance chromatography with electrochemical detection (HPLC-ED) techniques for the analysis of various drugs in biological samples for TDM have been described less often. Before chromatographic analysis, samples were pretreated by various procedures-most often by protein precipitation, liquid-liquid extraction, and solid-phase extraction, rarely by microextraction by packed sorbent, dispersive liquid-liquid microextraction. The aim of this article is to review the recent literature (2010-2020) regarding the use of liquid chromatography with various detection techniques for TDM.
    Keywords:  biological samples; detection techniques; liquid chromatography; sample preparation; therapeutic drug monitoring (TDM)
    DOI:  https://doi.org/10.3390/molecules25174026
  8. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Aug 27. pii: S1570-0232(20)30930-2. [Epub ahead of print]1159 122337
    Loh GOK, Wong EYL, Tan YTF, Lee YL, Pang LH, Chin MC, Damenthi N, Peh KK.
      A simple, rapid, sensitive, and reproducible liquid chromatography-tandem mass spectrometry method was developed to determine sitagliptin in human plasma. Diphenhydramine HCl was used as internal standard (IS). The chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 - Fast LC column (100 × 2.1mmID, 2.7) fitted with UHPLC Guard Poroshell 120 EC-C18 (5 × 2.1mmID, 2.7 µm). The mobile phase consisted of 0.1% v/v formic acid and methanol (45:55, v/v) run at a flow rate of 0.45 mL/min at 30 °C. Methanol produced relatively cleaner plasma sample as deproteinization agent. Polytetrafluoroethylene membrane was preferred over nylon membrane as the former produced clear plasma samples. The standard calibration curve was linear over the concentration range of 5-500.03 ng/mL. The within-run precision was 0.53-7.12% and accuracy 87.09-105.05%. The between-run precision was 4.74-11.68% and accuracy 95.02-97.36%. The extended run precision was 3.60-6.88% and accuracy 93.18-95.82%. The recovery of analyte and IS was consistent. Sitagliptin in plasma was stable at benchtop (short term) for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h (post-preparative), after 7 freeze-thaw cycles (-20 ± 10 °C), and 62 days in the freezer (-20 ± 10 °C). Both sitagliptin (analyte) and IS stock solutions were stable for 62 days when kept at room temperature (25 ± 4 °C) and in chiller (2-8 °C). The validated method was successfully applied to a bioequivalence study of two sitagliptin formulations involving 26 healthy Malaysian volunteers.
    Keywords:  Bioequivalence study; Polytetrafluoroethylene membrane; Protein precipitation; Sitagliptin
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122337
  9. Molecules. 2020 Sep 09. pii: E4116. [Epub ahead of print]25(18):
    Skubic C, Vovk I, Rozman D, Križman M.
      We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.
    Keywords:  LC-MS; PFP; cholesterol; cholesterol synthesis; pentafluorophenyl; sterol intermediates
    DOI:  https://doi.org/10.3390/molecules25184116
  10. Anal Methods. 2020 Sep 09.
    Sanders KL, Edwards JL.
      Liquid chromatography coupled to mass spectrometry (LC-MS) is one of the most powerful tools in identifying and quantitating molecular species. Decreasing column diameter from the millimeter to micrometer scale is now a well-developed method which allows for sample limited analysis. Specific fabrication of capillary columns is required for proper implementation and optimization when working in the nanoflow regime. Coupling the capillary column to the mass spectrometer for electrospray ionization (ESI) requires reduction of the subsequent emitter tip. Reduction of column diameter to capillary scale can produce improved chromatographic efficiency and the reduction of emitter tip size increased sensitivity of the electrospray process. This improved sensitivity and ionization efficiency is valuable in analysis of precious biological samples where analytes vary in size, ion affinity, and concentration. In this review we will discuss common approaches and challenges in implementing nLC-MS methods and how the advantages can be leveraged to investigate a wide range of biomolecules.
    DOI:  https://doi.org/10.1039/d0ay01194k
  11. Clin Chem Lab Med. 2020 Sep 11. pii: /j/cclm.ahead-of-print/cclm-2020-0610/cclm-2020-0610.xml. [Epub ahead of print]
    Austin D, John C, Hunt BJ, Carling RS.
      Objectives Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described. Methods Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 μL/min. The analysis time was 8 min. Results For each analyte linear calibration curves were obtained over the concentration range 50-2000 μg/L, the lower limit of quantification (LLOQ) was 13 μg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 μg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively. Conclusion This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.
    Keywords:  human whole blood; hydroxychloroquine; liquid chromatography tandem mass spectrometry; method validation
    DOI:  https://doi.org/10.1515/cclm-2020-0610