bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒07‒26
fifteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory


  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Jul 14. pii: S1570-0232(20)30302-0. [Epub ahead of print]1152 122266
    Lv W, Guo L, Zheng F, Wang Q, Wang W, Cui L, Ouyang Y, Liu X, Li E, Shi X, Xu G.
      Because of the greatly different physicochemical properties of metabolites, comprehensive metabolite profiling analysis has always been a challenging task. Reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) have been used to the analysis of nonpolar metabolites and polar metabolites, respectively. In this work, an alternate HILIC/RPLC-mass spectrometry (MS) approach was developed for the comprehensive and high-throughput analysis of polar and nonpolar metabolites. HILIC and RPLC are respectively performed on two ultra-high performance LC (UHPLC) systems, and coupled to one mass spectrometer to acquire the data. When HILIC gradient elution is running RPLC is in a washing and equilibration state, and vice versa. As a result, the total analysis time was reduced by about one third to 25.4 min. Two hundred and eight representative standards including at least twelve types of commonly met metabolites, SRM 1950 plasma, serum, urine and liver tissue samples were used to test the established alternate HILIC/RPLC-MS method. The results demonstrated that the method possessed high metabolite coverage. The developed method was validated to have good linearity and repeatability. As an example of application, 61 significantly changed metabolites in the colon cancer tissues were defined by this established method.
    Keywords:  Alternate separation; Colon cancer; Hydrophilic interaction liquid chromatography; Metabolite coverage; Reversed-phase liquid chromatography
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122266
  2. Metabolites. 2020 Jul 17. pii: E292. [Epub ahead of print]10(7):
    Sun X, Berger RS, Heinrich P, Marchiq I, Pouyssegur J, Renner K, Oefner PJ, Dettmer K.
      Glutathione (GSH) and glutathione disulfide (GSSG) are commonly used to assess the oxidative status of a biological system. Various protocols are available for the analysis of GSH and GSSG in biomedical specimens. In this study, we present an optimized protocol for the in situ derivatization of GSH with N-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio during sample preparation. The protocol comprises the incubation of cells in NEM containing phosphate buffered saline (PBS), followed by metabolite extraction with 80% methanol. Further, to preserve the use of QTOF-MS, which may lack the linear dynamic range required for the simultaneous quantification of GSH and GSSG in non-targeted metabolomics, we combined liquid chromatographic separation with the online monitoring of UV absorbance of GS-NEM at 210 nm and the detection of GSSG and its corresponding stable isotope-labeled internal standard by QTOF-MS operated with a 10 Da Q1 window. The limit of detection (LOD) for GS-NEM was 7.81 µM and the linear range extended from 15.63 µM to 1000 µM with a squared correlation coefficient R2 of 0.9997. The LOD for GSSG was 0.001 µM, and the lower limit of quantification (LLOQ) was 0.01 µM, with the linear (R2 = 0.9994) range extending up to 10 µM. The method showed high repeatability with intra-run and inter-run coefficients of variation of 3.48% and 2.51% for GS-NEM, and 3.11% and 3.66% for GSSG, respectively. Mean recoveries of three different spike-in levels (low, medium, high) of GSSG and GS-NEM were above 92%. Finally, the method was applied to the determination of changes in the GSH/GSSG ratio either in response to oxidative stress in cells lacking one or both monocarboxylate transporters MCT1 and MCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genes IDH1 and IDH2.
    Keywords:  UV detection; cell culture; glutathione; glutathione disulfide; liquid chromatography; mass spectrometry
    DOI:  https://doi.org/10.3390/metabo10070292
  3. Molecules. 2020 Jul 17. pii: E3258. [Epub ahead of print]25(14):
    Pautova A, Khesina Z, Getsina M, Sobolev P, Revelsky A, Beloborodova N.
      Indole-containing acids-tryptophan metabolites-found in serum and cerebrospinal fluid (CSF) samples of patients with diseases of the central nervous system (CNS) were determined with the use of microextraction by packed sorbent (MEPS) followed by silylation and gas chromatography-mass spectrometry (GC-MS) analysis. MEPS with the following silylation led to the reproducible formation of derivatives with an unsubstituted hydrogen ion in the indole ring, the chromatographic peaks of which are symmetric and can be used for GC-MS analysis without additional derivatization. The recoveries of analytes at the limit of quantitation (LOQ) levels were 40-80% for pooled CSF and 40-60% for serum. The limit of detection (LOD) and LOQ values were 0.2-0.4 and 0.4-0.5 µM, respectively, for both CSF and serum. The precision (the reproducibility, RSD) value of less than 20% and the accuracy (the relative error, RE) value of less than ±20% at the LOQ concentrations meet the Food and Drug Administration (FDA) recommendations. Linear correlations for all analytes were determined over a potentially clinically significant range of concentrations (0.4-10 µM for serum, R2 ≥ 0.9942, and 0.4-7 µM for CSF, R2 ≥ 0.9949). Moreover, MEPS significantly reduced the matrix effect of serum compared to liquid-liquid extraction (LLE), which was revealed in the example of reducing the amount of cholesterol and its relative compounds.
    Keywords:  5-hydroxyindole-3-acetic acid; gas chromatography–mass spectrometry; indole-3-acetic acid; indole-3-lactic acid; indole-3-propionic acid; microextraction by packed sorbent; silylation; tryptophan metabolites
    DOI:  https://doi.org/10.3390/molecules25143258
  4. Metabolites. 2020 Jul 17. pii: E296. [Epub ahead of print]10(7):
    Jenkins B, Ronis M, Koulman A.
      Typical lipidomics methods incorporate a liquid-liquid extraction with LC-MS quantitation; however, the classic sample extraction methods are not high-throughput and do not perform well at extracting the full range of lipids especially, the relatively polar species (e.g., acyl-carnitines and glycosphingolipids). In this manuscript, we present a novel sample extraction protocol, which produces a single phase supernatant suitable for high-throughput applications that offers greater performance in extracting lipids across the full spectrum of species. We applied this lipidomics pipeline to a ruminant fat dose-response study to initially compare and validate the different extraction protocols but also to investigate complex lipid biomarkers of ruminant fat intake (adjoining onto simple odd chain fatty acid correlations). We have found 100 lipids species with a strong correlation with ruminant fat intake. This novel sample extraction along with the LC-MS pipeline have shown to be sensitive, robust and hugely informative (>450 lipids species semi-quantified): with a sample preparation throughput of over 100 tissue samples per day and an estimated ~1000 biological fluid samples per day. Thus, this work facilitating both the epidemiological involvement of ruminant fat, research into odd chain lipids and also streamlining the field of lipidomics (both by sample preparation methods and data presentation).
    Keywords:  Folch; lipid profiling; odd chain lipids; protein precipitation; relative lipid composition (Mol%); sample preparation
    DOI:  https://doi.org/10.3390/metabo10070296
  5. Anal Chem. 2020 Jul 22.
    Shi X, Xi B, Jasbi P, Turner C, Jin Y, Gu H.
      Metabolic flux analysis (MFA) is highly relevant to understanding metabolic mechanisms of various biological processes. While the pace of methodology development in MFA has been rapid, a major challenge the field continues to witness is limited metabolite coverage, often restricted to a small to moderate number of well-known compounds. In addition, isotopic peaks from an enriched metabolite tend to have low abundances, which makes liquid chromatography tandem mass spectrometry (LC-MS/MS) highly useful in MFA due to its high sensitivity and specificity. Previously we have built large-scale LC-MS/MS approaches that can be routinely used for measurement of up to ~1,900 metabolite/feature levels [Gu et al. Anal. Chem. 2015, 87, 12355-62; Shi et al. Anal. Chem. 2019, 91,13737-45]. In this study, we aim to expand our previous studies focused on metabolite level measurements to flux analysis and establish a novel comprehensive isotopic targeted mass spectrometry (CIT-MS) method for reliable MFA analysis with broad coverage. As a proof-of-principle, we have applied CIT-MS to compare the steady-state enrichment of metabolites between Myc(oncogene)-On and Myc-Off Tet21N human neuroblastoma cells cultured with U-13C6-glucose medium. CIT-MS is operationalized using multiple reaction monitoring (MRM) mode and is able to perform MFA of 310 identified metabolites (142 reliably detected, 46 kinetically profiled) selected from >35 metabolic pathways of strong biological significance. Further, we developed a novel concept of relative flux, which eliminates the requirement of absolute quantitation in traditional MFA and thus enables comparative MFA under the pseudosteady state. As a result, CIT-MS was shown to possess the advantages of broad coverage, easy implementation, fast throughput, and more importantly, high fidelity and accuracy in MFA. In principle, CIT-MS can be easily adapted to track the flux of other labeled tracers (such as 15N-tracers) in any metabolite detectable by LC-MS/MS and in various biological models (such as mice). Therefore, CIT-MS has great potential to bring new insights to both basic and clinical metabolism research.
    DOI:  https://doi.org/10.1021/acs.analchem.0c01767
  6. Biomed Chromatogr. 2020 Jul 24. e4951
    Öztürk Er E, Şahin A, Bakırdere S.
      Quantitative determination of omega-6 and omega-3 polyunsaturated fatty acids in human plasma and urine with high accuracy and precision provides significant information to monitor the underlying etiology of several diseases. In this regard, liquid chromatography-mass spectrometry is a good choice owing to its great selectivity and sensitivity. Additionally, the hybrid quadrupole-time of flight - mass spectrometer systems provides easy identification of target compounds with superior mass measurements. In this study, an analytical method has been developed for simple, accurate and simultaneous determination of linoleic acid, arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid in a short chromatographic analysis period. The developed method is suitable for the quantitative detection of these four compounds with detection limits ranging between 1.1 - 3.0 ng mL-1 and its applicability was assessed in human urine and plasma samples. As a result, acceptable accuracy (between 83-111%) and good precision (<6%) were obtained for target compounds using matrix matching calibration strategy.
    Keywords:  Polyunsaturated fatty acids; human plasma; human urine; liquid chromatography - mass spectrometry; matrix matching calibration strategy
    DOI:  https://doi.org/10.1002/bmc.4951
  7. Ther Drug Monit. 2020 Jul 16.
    de Jager NCB, Heijdra JM, Pistorius M, Kruip MJHA, Leebeek FWG, Cnossen MH, Mathôt RAA.
      BACKGROUND: Desmopressin (D-amino D-arginine vasopressin: dDAVP) is used for the treatment of patients with hemophilia A and Von Willebrand disease. Studies on the rationale of dosing are scarce and mainly focus on the underlying causes of the vast differences in desmopressin response among individuals. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of desmopressin in human plasma for identifying its pharmacokinetics and its therapeutic effect relationship in patients with bleeding disorder.METHODS: The method entails solid-phase extraction with ion exchange for sample clean-up, followed by an LC-MS/MS run. The technique has been validated for analytical selectivity as well as specificity, process efficiency, linearity, accuracy, imprecision, and stability.
    RESULTS: This method showed good selectivity as no significant chromatographic matrix interferences were observed. The determination coefficient (R) of the calibration curves was ≥ 0.990. Analyte accuracy ranged from 89.2% to 111.8%, and the between- and within-run imprecision was less than 9.3% in a plasma concentration range from 60 to 3200 pg/mL. Samples were stable during three freeze/thaw cycles with an additional 120 hours of storage at room temperature (21°C - 24°C) and 96 hours in the auto-sampler (10°C). The total run time was approximately 5 minutes.
    CONCLUSIONS: The LC-MS/MS method presented enables quantification of desmopressin in human plasma, and it is sensitive, specific, efficient, accurate, and precise. This analytical technique is a valuable and useful tool to study the inter-patient variability of pharmacokinetics.
    DOI:  https://doi.org/10.1097/FTD.0000000000000791
  8. Wei Sheng Yan Jiu. 2020 May;49(3): 447-457
    Li S, Fu Z, Zhang H, Hu Y, Wang R, Chen J, Mao D, Liu Z, Wang J, Yang L.
      OBJECTIVE: Comparison of liquid chromatography-tandem mass spectrometry(LC-MS/MS) and enzyme-linked immunosorbent assay(ELISA) for the detection of 25(OH)D concentration in human serum and the diagnosis of vitamin D deficiency.METHODS: In the serum pool of &quot;National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention&quot;, 500 serum samples of women aged 18 to 45 years old were randomly selected, and 25(OH)D levels were measured by ELISA and LC-MS/MS for the same serum sample, respectively. The LC-MS/MS column was Waters XBridge BEH C_(18)(2. 1 mm×50 mm, 2. 5 μm). The correlation between the two method was tested by correlation analysis, regression analysis and consistency test. The Endocrine Society and Institute of Medicine recommendations were used to determine the deficiency of vitamin D, and the McNemar test, Kappa coefficient and diagnostic test were used to diagnose the consistency of vitamin D deficiency.
    RESULTS: The regression equation for the 25(OH)D concentration measured by the two method was y_(LC-MS/MS)=-0. 035+1. 007×x_(ELISA)(r=0. 877), and the average deviation between the two was 4. 48% and the intraclass correlation coefficient was 0. 87. The 25(OH)D concentration was less than 12 and 20 ng/mL as a criterion for vitamin D deficiency, and the Kappa coefficients were greater than 0. 60(0. 64 and 0. 74).
    CONCLUSION: When serum 25(OH)D level was detected by LC-MS/MS and ELISA, the correspondence of the two method was fine. Taking the "gold standard" LC-MS/MS method as a reference, the ELISA method was used to determine human vitamin D deficiency with good sensitivity and specificity, which can be used for the accurate and rapid detection of large-scale population samples.
    Keywords:  25(OH)D; blood test; enzyme-linked immunosorbent assay; liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10. 19813/j. cnki. weishengyanjiu. 2020. 03. 017
  9. J Chromatogr A. 2020 Aug 16. pii: S0021-9673(20)30559-8. [Epub ahead of print]1625 461281
    Pérez-Baeza M, Escuder-Gilabert L, Martín-Biosca Y, Sagrado S, Medina-Hernández MJ.
      Polysaccharide-based chiral stationary phases (CSPs) are the most used chiral selectors in HPLC. These CSPs can be used in normal, polar organic and aqueous-organic mobile phases. However, normal and polar organic mobile phases are not adequate for chiral separation of polar compounds, for the analysis of aqueous samples and for MS detection. In these situations, reversed phase conditions, without the usual non-volatile additives incompatible with MS detection, are preferable. Moreover, in most of the reported chiral chromatographic methods, retention is too large for routine work. In this paper, the chiral separation of 53 structurally unrelated compounds is studied using three commercial amylose-based CSPs -coated amylose tris(3,5-dimethylphenylcarbamate) (Am1), coated amylose tris(5-chloro-2-methylphenylcarbamate) (Am2), and immobilised amylose tris(3-chloro-5-methylphenylcarbamate) (Am3)-. Chiral separations are carried out using acetonitrile/ammonium bicarbonate (pH = 8.0) mixtures, reversed mobile phases compatible with MS detection. To provide realistic conditions for routine analysis, maximum retention factors are set to 15. Retention and enantioresolution behaviour of compounds in those CSPs are compared. On the other hand, to compare and describe the resolution ability of these CSPs, 58 structural variables of the compounds are tested to model for the first time a categorical enantioresolution (CRs) for Am1 and Am3 CSPs. Discriminant partial least squares, for one response categorical variable (DPLS1) is used for feature selection, modelling. The final DPLS1 models showed good descriptive ability.
    Keywords:  Amylose-based chiral stationary phases. Reversed phase liquid chromatography. Enantioresolution modelling and description. Discriminant partial least squares. Feature selection
    DOI:  https://doi.org/10.1016/j.chroma.2020.461281
  10. Biomolecules. 2020 Jul 22. pii: E1092. [Epub ahead of print]10(8):
    Kokotou MG.
      Fatty acid esters of hydroxy fatty acids (FAHFAs) constitute a class of recently identified novel lipids exhibiting anti-diabetic and anti-inflammatory effects. Due to their high biological significance, a tremendous effort has been devoted to the development of analytical methods for the detection and quantitation of FAHFAs during the last five years. The analysis of FAHFAs is very challenging due to the great number of possible regio-isomers arising from the great number of possible combinations of FAs with HFAs, and the low abundancies of FAHFAs in biological samples. The aim of this review article is to summarize all the cutting-edge analytical methodologies for the determination of FAHFAs in biological samples, plant tissues and food matrices, with emphasis on extraction and analysis steps. All the analytical methodologies rely on the use of liquid chromatography-mass spectrometry (LC-MS), providing high sensitivity due to the MS detection. Powerful and robust analytical methodologies may highly contribute in studying FAHFAs levels under various biomedical conditions, and facilitate our understanding of the role of these lipid species in physiological and pathological conditions.
    Keywords:  anti-diabetic; anti-inflammatory; fatty acid esters of hydroxy fatty acids (FAHFAs); lipidomics; liquid chromatography–mass spectrometry (LC-MS)
    DOI:  https://doi.org/10.3390/biom10081092
  11. Anal Bioanal Chem. 2020 Jul 22.
    Hellhake S, Meckelmann SW, Empl MT, Rentmeister K, Wißdorf W, Steinberg P, Schmitz OJ, Benter T, Schebb NH.
      Eicosanoids and other oxylipins play an important role in mediating inflammation as well as other biological processes. For the investigation of their biological role(s), comprehensive analytical methods are necessary, which are able to provide reliable identification and quantification of these compounds in biological matrices. Using charge-switch derivatization with AMPP (N-(4-aminomethylphenyl)pyridinium chloride) in combination with liquid chromatography ion mobility quadrupole time-of-flight mass spectrometry (LC-IM-QTOF-MS), we developed a non-target approach to analyze oxylipins in plasma, serum, and cells. The developed workflow makes use of an ion mobility resolved fragmentation to pinpoint derivatized molecules based on the cleavage of AMPP, which yields two specific fragment ions. This allows a reliable identification of known and unknown eicosanoids and other oxylipins. We characterized the workflow using 52 different oxylipins and investigated their fragmentation patterns and ion mobilities. Limits of detection ranged between 0.2 and 10.0 nM (1.0-50 pg on column), which is comparable with other state-of-the-art methods using LC triple quadrupole (QqQ) MS. Moreover, we applied this strategy to analyze oxylipins in different biologically relevant matrices, as cultured cells, human plasma, and serum. Graphical abstract.
    Keywords:  AMPP; DTIMS; Eicosanoids; Ion mobility-mass spectrometry; Lipidomics; Oxylipins
    DOI:  https://doi.org/10.1007/s00216-020-02795-2
  12. J Chromatogr A. 2020 Aug 16. pii: S0021-9673(20)30556-2. [Epub ahead of print]1625 461278
    Ďurč P, Dosedělová V, Foret F, Dolina J, Konečný Š, Himmelsbach M, Buchberger W, Kubáň P.
      A fast, non-invasive, high-performance liquid chromatographic screening method with electrospray ionization mass spectrometric detection was developed for the analysis of three major glycine-conjugated bile acids in human saliva. Using a mobile phase composed of 80% methanol and 0.1% formic acid, glycocholic, glycodeoxycholic, and glycochenodeoxycholic acids were separated in less than 4 minutes with sensitivity in the low nM range. Bile acids are thought to contribute to the pathology of various complications in gastroesophageal reflux disease, for instance, Barrett's esophagus, which may eventually lead to esophageal carcinoma. In this pilot study, samples of saliva obtained from 15 patients with Barrett's esophagus of various severities were compared to saliva samples from 10 healthy volunteers. Glycochenodeoxycholic acid was significantly elevated in the patients and principal component analysis of all bile acids could distinguish the most severe Barrett's esophagus patients. We also reported on the detection of glycochenodeoxycholic acid in exhaled breath condensate for the first time. The promising results of this pilot study warrant future investigation, aiming at non-invasive diagnostics of Barrett's esophagus susceptibility in patients with gastroesophageal reflux disease.
    Keywords:  Barrett's esophagus; Bile acids; HPLC-MS; Saliva
    DOI:  https://doi.org/10.1016/j.chroma.2020.461278
  13. Metabolites. 2020 Jul 17. pii: E295. [Epub ahead of print]10(7):
    Torell F, Skotare T, Trygg J.
      Data integration has been proven to provide valuable information. The information extracted using data integration in the form of multiblock analysis can pinpoint both common and unique trends in the different blocks. When working with small multiblock datasets the number of possible integration methods is drastically reduced. To investigate the application of multiblock analysis in cases where one has a few number of samples and a lack of statistical power, we studied a small metabolomic multiblock dataset containing six blocks (i.e., tissue types), only including common metabolites. We used a single model multiblock analysis method called the joint and unique multiblock analysis (JUMBA) and compared it to a commonly used method, concatenated principal component analysis (PCA). These methods were used to detect trends in the dataset and identify underlying factors responsible for metabolic variations. Using JUMBA, we were able to interpret the extracted components and link them to relevant biological properties. JUMBA shows how the observations are related to one another, the stability of these relationships, and to what extent each of the blocks contribute to the components. These results indicate that multiblock methods can be useful even with a small number of samples.
    Keywords:  OnPLS; data integration; joint and unique multiblock analysis (JUMBA); metabolomics; multi-tissue; multiblock; multiblock orthogonal component analysis (MOCA)
    DOI:  https://doi.org/10.3390/metabo10070295
  14. Anal Methods. 2020 Jul 23.
    Caban M, Stepnowski P.
      The study aimed to show the limitations and advantages of the use of stable isotope labeled internal standards (SILISs) for the quantification of small polar compounds (ibuprofen, diclofenac, metoprolol, bisphenol A, 17β-estradiol) in water samples by GC/MS with selected ion monitoring (SIM). The selection of SIM ions shows that for some of the analytes, in the form of trimethylsilylated derivatives, it is problematic to have a minimum of two qualifiers because of the poor mass spectra, high impact of the background or overlapping of the peaks from the analytes and SILISs. The "isotope effect" was observed on GC chromatograms. During the development of the SIM method, special attention was given to the qualifier/quantifier ratio, for which a variety of acceptance criteria can be found in the official guidelines. The values of the solid-phase extraction efficiency, as well as the matrix effect and absolute recovery were the same for the analytes and their corresponding SILISs. The developed method was used for the quantification of analytes in wastewaters.
    DOI:  https://doi.org/10.1039/d0ay00723d
  15. Anal Methods. 2020 Jul 28. 12(28): 3582-3591
    Plyushchenko I, Shakhmatov D, Bolotnik T, Baygildiev T, Nesterenko PN, Rodin I.
      The data processing workflow for LC-MS based metabolomics study is suggested with signal drift correction, univariate analysis, supervised learning, feature selection and unsupervised modelling. The proposed approach requires only an annotation-free peak table and produces an extremely reduced set of the most relevant features together with validation via Receiver Operating Characteristic analysis for selected predictors, cross-validation and unsupervised projection. The presented study was initially optimised by its own experimental set and then was successfully tested by using 36 datasets from 21 publicly available metabolomics projects. The suggested workflow can be used for classification purposes in high dimensional metabolomics studies and as a first step in exploratory analysis, data projection, biomarker selection, data integration and fusion.
    DOI:  https://doi.org/10.1039/d0ay00204f