bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒11‒08
seventeen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory


  1. Anal Chim Acta. 2020 Nov 15. pii: S0003-2670(20)30908-9. [Epub ahead of print]1137 74-84
    Wolrab D, Chocholoušková M, Jirásko R, Peterka O, Mužáková V, Študentová H, Melichar B, Holčapek M.
      Effects of blood collection tubes, the time period, the sample origin, and the method used on the lipidomic profile are investigated by ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS) and hydrophilic interaction liquid chromatography ultrahigh-performance liquid chromatography - mass spectrometry (HILIC-UHPLC/MS). Heparin plasma samples have been obtained from 99 healthy volunteers at three time points separated by six-month intervals together with one collection for EDTA plasma and serum. Furthermore, lipid concentrations in heparin plasma collected at two different sites are compared. 171 lipid species from eight lipid classes are quantified with UHPSFC/MS, and 122 lipid species from four lipid classes with HILIC-UHPLC/MS. The accuracy of both methods is monitored by the quantitation error using two internal standards (IS) per individual lipid classes. No significant differences in lipid profiles are observed for different time points and types of collection tubes (heparin plasma, EDTA plasma, and serum). Most pronounced lipid concentration differences are observed for the comparison of NIST SRM 1950 human plasma and mean lipid concentrations of the investigated cohort. Furthermore, differences in lipid concentrations are observed between employed methods (UHPSFC/MS vs. HILIC-UHPLC/MS), which can be compensated by the normalization using NIST SRM 1950 human plasma used as the quality control sample.
    Keywords:  Blood collection; Human plasma; Lipidomics; Lipids; Liquid chromatography; Mass spectrometry; Serum; Supercritical fluid chromatography
    DOI:  https://doi.org/10.1016/j.aca.2020.08.061
  2. Anal Chim Acta. 2020 Nov 15. pii: S0003-2670(20)30912-0. [Epub ahead of print]1137 37-46
    Guo J, Huan T.
      Despite the growing popularity of liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, no study has yet to systematically compare the performance of different data acquisition modes in the discovery of significantly altered metabolic features, which is an important task of untargeted metabolomics for identifying clinical biomarkers and elucidating disease mechanism in comparative samples. In this work, we performed a comprehensive comparison of three most commonly used data acquisition modes, including full-scan, data-dependent acquisition (DDA), and data-independent acquisition (DIA), using a metabolomics study of human plasma samples from leukemia patients before and after one-month chemotherapy. After optimization of data processing parameters, we extracted and compared statistically significant metabolic features from the results of each data acquisition mode. We found that most significant features can be consistently found in all three data acquisition modes with similar statistical performance as evaluated by Pearson correlation and receiver operating characteristic (ROC) analysis. Upon comparison, DDA mode consistently generated fewer uniquely found significant features than full-scan and DIA modes. We then manually inspected over 2000 uniquely discovered significant features in each data acquisition mode and showed that these features can be generally categorized into four major types. Many significant features were missed in DDA mode, primarily due to its low capability of detecting or extracting these features from raw LC-MS data. We thus proposed a bioinformatic solution to rescue these missing significant features from the raw DDA data with good reproducibility and accuracy. Overall, our work asserts that data acquisition modes can influence metabolomics results, suggesting room for improvement of data acquisition modes for untargeted metabolomics.
    Keywords:  Data acquisition mode; Data-dependent acquisition; Data-independent acquisition; Full-scan; Liquid chromatography-mass spectrometry; Untargeted metabolomics
    DOI:  https://doi.org/10.1016/j.aca.2020.08.065
  3. Anal Chim Acta. 2020 Nov 15. pii: S0003-2670(20)30993-4. [Epub ahead of print]1137 156-169
    Xu T, Hu C, Xuan Q, Xu G.
      Lipids are vital biological molecules and play multiple roles in cellular function of mammalian organisms such as cellular membrane anchoring, signal transduction, material trafficking and energy storage. Driven by the biological significance of lipids, lipidomics has become an emerging science in the field of omics. Lipidome in biological systems consists of hundreds of thousands of individual lipid molecules that possess complex structures, multiple categories, and diverse physicochemical properties assembled by different combinations of polar headgroups and hydrophobic fatty acyl chains. Such structural complexity poses a huge challenge for comprehensive lipidome analysis. Thanks to the great innovations in chromatographic separation techniques and the continuous advances in mass spectrometric detection tools, analytical strategies for lipidomics have been highly diversified so that the depth and breadth of lipidomics have been greatly enhanced. This review will present the current state of mass spectrometry-based analytical strategies including untargeted, targeted and pseudotargeted lipidomics. Recent typical applications of lipidomics in biomarker discovery, pathogenic mechanism and therapeutic strategy are summarized, and the challenges facing to the field of lipidomics are also discussed.
    Keywords:  Biomarker discovery; High-resolution mass spectrometry; Liquid chromatography; Pseudotargeted lipidomics; Targeted lipidomics; Untargeted lipidomics
    DOI:  https://doi.org/10.1016/j.aca.2020.09.060
  4. Anal Bioanal Chem. 2020 Nov 06.
    Aghai F, Zimmermann S, Kurlbaum M, Jung P, Pelzer T, Klinker H, Isberner N, Scherf-Clavel O.
      A liquid chromatography tandem mass spectrometry method for the analysis of ten kinase inhibitors (afatinib, axitinib, bosutinib, cabozantinib, dabrafenib, lenvatinib, nilotinib, osimertinib, ruxolitinib, and trametinib) in human serum and plasma for the application in daily clinical routine has been developed and validated according to the US Food and Drug Administration and European Medicines Agency validation guidelines for bioanalytical methods. After protein precipitation of plasma samples with acetonitrile, chromatographic separation was performed at ambient temperature using a Waters XBridge® Phenyl 3.5 μm (2.1 × 50 mm) column. The mobile phases consisted of water-methanol (9:1, v/v) with 10 mM ammonium bicarbonate as phase A and methanol-water (9:1, v/v) with 10 mM ammonium bicarbonate as phase B. Gradient elution was applied at a flow rate of 400 μL/min. Analytes were detected and quantified using multiple reaction monitoring in electrospray ionization positive mode. Stable isotopically labeled compounds of each kinase inhibitor were used as internal standards. The acquisition time was 7.0 min per run. All analytes and internal standards eluted within 3.0 min. The calibration curves were linear over the range of 2-500 ng/mL for afatinib, axitinib, bosutinib, lenvatinib, ruxolitinib, and trametinib, and 6-1500 ng/mL for cabozantinib, dabrafenib, nilotinib, and osimertinib (coefficients of correlation ≥ 0.99). Validation assays for accuracy and precision, matrix effect, recovery, carryover, and stability were appropriate according to regulatory agencies. The rapid and sensitive assay ensures high throughput and was successfully applied to monitor concentrations of kinase inhibitors in patients. Graphical abstract.
    Keywords:  Afatinib; Kinase inhibitors; Liquid chromatography tandem mass spectrometry (LC-MS/MS); Osimertinib; Therapeutic drug monitoring
    DOI:  https://doi.org/10.1007/s00216-020-03031-7
  5. Anal Chem. 2020 Nov 05.
    Weng CY, Kuo TH, Chai LMX, Zou HB, Feng TH, Huang YJ, Tsai JC, Wu PH, Chiu YW, Lan EI, Sheen LY, Hsu CC.
      Short-chain fatty acids (SCFAs) are small molecules ubiquitous in nature. In mammalian guts, SCFAs are mostly produced by anaerobic intestinal microbiota through the fermentation of dietary fiber. Levels of microbe-derived SCFAs are closely relevant to human health status and indicative to gut microbiota dysbiosis. However, the quantification of SCFA using conventional chromatographic approaches is often time consuming, thus limiting high-throughput screening tests. Herein, we established a novel method to quantify SCFAs by coupling amidation derivatization of SCFAs with paper-loaded direct analysis in real time mass spectrometry (pDART-MS). Remarkably, SCFAs of a biological sample were quantitatively determined within a minute using the pDART-MS platform, which showed a limit of detection at the μM level. This platform was applied to quantify SCFAs in various biological samples, including feces from stressed rats, sera of patients with kidney disease, and fermentation products of metabolically engineered cyanobacteria. Significant differences in SCFA levels between different groups of biological practices were promptly revealed and evaluated. As there is a burgeoning demand for the analysis of SCFAs due to an increasing academic interest of gut microbiota and its metabolism, this newly developed platform will be of great potential in biological and clinical sciences as well as in industrial quality control.
    DOI:  https://doi.org/10.1021/acs.analchem.0c03862
  6. Data Brief. 2020 Dec;33 106382
    Borah K, Rickman OJ, Voutsina N, Baple EL, Dias IH, Crosby AH, Griffiths HR.
      The raw datasets of oxysterol quantifications from whole cell and mitochondrial fractions of THP-1 monocytes and macrophages, neuronal-like SH-SH5Y cells and human peripheral blood mononuclear cells are presented. Oxysterols were quantified using a new liquid chromatography-mass spectrometry (LC-MS) and multiple reaction monitoring analysis published in the article "A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism" in Redox Biology [1]. This method showed improved extraction efficiency and recovery of mono and dihydroxycholesterols from cellular matrix. The datasets derived from the three cell lines are included in the appendix. These datasets provide new information about the oxysterol distribution in THP-1 monocytes and macrophages, SH-SY5Y cells and peripheral blood mononuclear cells. These datasets can be used as a guide for oxysterol distribution in the three cell lines for future studies, and can used for future method optimization, and for comparison of oxysterol recovery with other analytical techniques.
    Keywords:  Cholesterol; Liquid chromatography-mass spectrometry; Metabolism; Mitochondria; Oxysterol; Peripheral blood mononuclear cell
    DOI:  https://doi.org/10.1016/j.dib.2020.106382
  7. Anal Chim Acta. 2020 Nov 15. pii: S0003-2670(20)30911-9. [Epub ahead of print]1137 64-73
    Pecher D, Zelinkova Z, Lucenicova J, Peppelenbosch M, Dokupilova S, Mikusova V, Mikus P.
      Thiopurine (TP) treatment is discontinued in up to 30% of patients suffering from inflammatory bowel diseases (IBD) due to various adverse effects. Therapeutic drug monitoring of biologically active TP metabolites, i.e. thiopurine nucleotides (TPN), can help to optimize the efficacy and safety of the TP treatment. In our work, a novel strategy for TPN profiling, based on a porous graphitic carbon (PGC) chromatography, was developed. The validated PGC-MS method was compared with ion-exchange LC-MS, a currently leading analytical approach established for the determination of TPN. The innovative approach enabled an enhancement of several key performance parameters demanded in a clinical routine use, namely (i) selectivity (time- and mass-recognition of all 12 TPN in one run), (ii) sensitivity (2-5-fold increase in intensities of the analytical signals), (iii) sample throughput (25% shorter analysis time). Application of the novel TPN profiling strategy to a pilot clinical study (12 patients) revealed significantly higher levels of 6-methylthioguanine 5'-diphosphate (MeTGDP) in non-responsive IDB patients treated with azathioprine. Some other TPN are very close to the critical level (p = 0.05) and they will need larger groups of IBD patients to confirm definitively their relevance. In conclusion, the developed PGC-MS method represents a significant improvement to currently available methods for detailed profiling of TPN and its use in bigger clinical studies should lead to a better understanding of the relationship between TPN profiles and therapeutic outcome.
    Keywords:  Azathioprine; Inflammatory bowel diseases; Mass spectrometry; Porous graphitic carbon based chromatography; Therapeutic drug monitoring; Thiopurine nucleotides
    DOI:  https://doi.org/10.1016/j.aca.2020.08.064
  8. Anal Chim Acta. 2020 Nov 15. pii: S0003-2670(20)30988-0. [Epub ahead of print]1137 136-142
    Benke PI, Burla B, Ekroos K, Wenk MR, Torta F.
      Contamination from the polymeric material released by vial caps used for sample introduction in liquid chromatography can significantly affect the signal of the analyte of interest. In particular, repeated injections from the same sample vial can enhance this suppressing effect. Multiple injections of the same sample are often used in metabolomics and lipidomics during routine analyses. Here we demonstrate how the presence of contaminant polymers, originating from the vial closures, significantly influences the estimation of the relative amount of endogenous lipids in human plasma. Furthermore, this can negatively impact other operations in mass spectrometric analysis, such as instrument equilibration and tuning or the common use of technical replicates to improve confidence in data interpretation. Our observations provide critical information on how to improve future analyses through the use of appropriate vial caps, solvents, chromatographic separations and equipment.
    Keywords:  Interferents; Lipidomics; Mass spectrometry; Polymers; Polysiloxanes; Vial septum
    DOI:  https://doi.org/10.1016/j.aca.2020.09.055
  9. Metabolites. 2020 Nov 02. pii: E443. [Epub ahead of print]10(11):
    Olesova D, Galba J, Piestansky J, Celusakova H, Repiska G, Babinska K, Ostatnikova D, Katina S, Kovac A.
      Autism spectrum disorder is a heterogeneous neurodevelopmental disease. Currently, no biomarker of this disease is known. Diagnosis is performed through observation, standardized behavioral scales, and interviews with parents. In practice, diagnosis is often delayed to the average age of four years or even more which adversely affects a child's perspective. A laboratory method allowing to detect the disorder at earlier stages is of a great need, as this could help the patients to start with treatment at a younger age, even prior to the clinical diagnosis. Recent evidence indicates that metabolomic markers should be considered as diagnostic markers, also serving for further differentiation and characterization of different subgroups of the autism spectrum. In this study, we developed an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry method for the simultaneous determination of six metabolites in human urine. These metabolites, namely methylguanidine, N-acetyl arginine, inosine, indole-3-acetic acid, indoxyl sulfate and xanthurenic acid were selected as potential biomarkers according to prior metabolomic studies. The analysis was carried out by means of reversed-phase liquid chromatography with gradient elution. Separation of the metabolites was performed on a Phenomenex Luna® Omega Polar C18 (100 × 1.0 mm, 1.6 µm) column at a flow rate of 0.15 mL/min with acetonitrile/water 0.1% formic acid aqueous as the mobile phase. The analysis was performed on a group of children with autism spectrum disorder and age-matched controls. In school children, we have detected disturbances in the levels of oxidative stress markers connected to arginine and purine metabolism, namely methylguanidine and N-acetylargine. Also, products of gut bacteria metabolism, namely indoxyl sulfate and indole-3-acetic acid, were found to be elevated in the patients' group. We can conclude that this newly developed method is fast, sensitive, reliable, and well suited for the quantification of proposed markers.
    Keywords:  autism spectrum disorder; gut microbiota; liquid chromatography; mass spectrometry; metabolomics; oxidative stress
    DOI:  https://doi.org/10.3390/metabo10110443
  10. Molecules. 2020 Nov 04. pii: E5128. [Epub ahead of print]25(21):
    Crook AA, Powers R.
      Nuclear Magnetic Resonance (NMR) spectroscopy is a quantitative analytical tool commonly utilized for metabolomics analysis. Quantitative NMR (qNMR) is a field of NMR spectroscopy dedicated to the measurement of analytes through signal intensity and its linear relationship with analyte concentration. Metabolomics-based NMR exploits this quantitative relationship to identify and measure biomarkers within complex biological samples such as serum, plasma, and urine. In this review of quantitative NMR-based metabolomics, the advancements and limitations of current techniques for metabolite quantification will be evaluated as well as the applications of qNMR in biomedical metabolomics. While qNMR is limited by sensitivity and dynamic range, the simple method development, minimal sample derivatization, and the simultaneous qualitative and quantitative information provide a unique landscape for biomedical metabolomics, which is not available to other techniques. Furthermore, the non-destructive nature of NMR-based metabolomics allows for multidimensional analysis of biomarkers that facilitates unambiguous assignment and quantification of metabolites in complex biofluids.
    Keywords:  biomarkers; metabolomics; multidimensional NMR; qNMR; quantitative NMR
    DOI:  https://doi.org/10.3390/molecules25215128
  11. Anal Chem. 2020 Nov 02.
    Li A, Conant CR, Zheng X, Bloodsworth KJ, Orton DJ, Garimella SVB, Attah IK, Nagy G, Smith RD, Ibrahim YM.
      The collision cross section (CCS) is an important property that aids in the structural characterization of molecules. Here, we investigated the CCS calibration accuracy with traveling wave ion mobility spectrometry (TWIMS) separations in structures for lossless ion manipulations (SLIM) using three sets of calibrants. A series of singly negatively charged phospholipids and bile acids were calibrated in nitrogen buffer gas using two different TW waveform profiles (square and sine) and amplitudes (20, 25, and 30 V0-p). The calibration errors for the three calibrant sets (Agilent tuning mixture, polyalanine, and one assembled in-house) showed negligible differences using a sine-shaped TW waveform. Calibration errors were all within 1-2% of the drift tube ion mobility spectrometry (DTIMS) measurements, with lower errors for sine waveforms, presumably due to the lower average and maximum fields experienced by ions. Finally, ultrahigh-resolution multipass (long path length) SLIM TWIMS separations demonstrated improved CCS calibration for phospholipid and bile acid isomers.
    DOI:  https://doi.org/10.1021/acs.analchem.0c02829
  12. Chempluschem. 2020 Nov;85(11): 2419-2427
    Pei C, Wan J.
      The efficient detection of small molecules is of significance for environmental monitoring, pharmacology, metabolomics, and lipidomics. The laser desorption/ionization mass spectrometry (LDI MS) platform enables high sensitivity, accuracy, resolution, and throughput in molecular analysis, but its analytical capability with respect to small molecules is limited due to inherent drawbacks arising from conventional organic matrices. The selection of an appropriate matrix is thus a precondition for small molecule detection by LDI MS. To date, various inorganic matrices have been developed, with a growing interest in composite materials displaying synergetic effects. This Minireview highlights the development of nanocomposites as LDI matrices driven by numerous innovations in material science, and their emerging use in small-molecule analysis.
    Keywords:  laser desorption ionization; mass spectrometry; metal-organic frameworks; nanocomposites; noble metals
    DOI:  https://doi.org/10.1002/cplu.202000619
  13. Metab Eng. 2020 Nov 03. pii: S1096-7176(20)30168-3. [Epub ahead of print]
    Antoniewicz MR.
      The field of metabolic engineering is primarily concerned with improving the biological production of value-added chemicals, fuels and pharmaceuticals through the design, construction and optimization of metabolic pathways, redirection of intracellular fluxes, and refinement of cellular properties relevant for industrial bioprocess implementation. Metabolic network models and metabolic fluxes are central concepts in metabolic engineering, as was emphasized in the first paper published in this journal, "Metabolic fluxes and metabolic engineering" (Metabolic Engineering, 1: 1-11, 1999). In the past two decades, a wide range of computational, analytical and experimental approaches have been developed to interrogate the capabilities of biological systems through analysis of metabolic network models using techniques such as flux balance analysis (FBA), and quantify metabolic fluxes using constrained-based modeling approaches such as metabolic flux analysis (MFA) and more advanced experimental techniques based on the use of stable-isotope tracers, i.e. 13C-metabolic flux analysis (13C-MFA). In this review, we describe the basic principles of metabolic flux analysis, discuss current best practices in flux quantification, highlight potential pitfalls and alternative approaches in the application of these tools, and give a broad overview of pragmatic applications of flux analysis in metabolic engineering practice.
    Keywords:  Flux balance analysis; Metabolic flux analysis; Metabolic network model; Metabolism; Stable-isotope tracers; Systems biology
    DOI:  https://doi.org/10.1016/j.ymben.2020.11.002
  14. J Chromatogr A. 2020 Oct 10. pii: S0021-9673(20)30880-3. [Epub ahead of print]1633 461606
    Petrarca MH, Braga PAC, Reyes FGR, Bragotto APA.
      An accurate, sensitive and selective analytical method is proposed for sulfonamide residues analysis in infant formulas based on hydrophilic interaction liquid chromatography (HILIC) and quadrupole time-of-flight mass spectrometry in full scan mode. The sample preparation approach involves low-temperature lipid precipitation followed by dispersive solid-phase extraction with PSA and C18 sorbents, which was successfully optimized using Plackett-Burman design. In order to achieve high analytical sensitivity, the influence of HILIC conditions on sulfonamide ionization was investigated, such as the mobile phase composition, buffer concentration, and sample diluent for injection. The method performance characteristics, including linearity (range 5-120 µg kg-1), reliable limits of quantification (between 5 and 20 µg kg-1), recovery (72.9-109.2%) and precision (coefficient of variation values ≤ 19.8%) under repeatability and within-laboratory reproducibility conditions, were in accordance with the Codex Alimentarius Commission CAC/GL 71-2009 for quantitative analytical methods for veterinary drug residues in foods. Moreover, adequate identification of the compounds was provided with accurate mass measurement of both precursor and fragment ions in one single run. Finally, the developed method was applied to thirty-five powdered milk-based infant formula samples available in the Brazilian market.
    Keywords:  Dispersive solid-phase extraction; HILIC; Low-temperature cleanup; Plackett-Burman design; QToF-MS; Veterinary drug residues
    DOI:  https://doi.org/10.1016/j.chroma.2020.461606
  15. J Adv Res. 2020 Nov;26 1-13
    Amer S, Zarad W, El-Gendy H, Abdel-Salam R, Hadad G, Emara S, Masujima T.
      Introduction: In recent years, analytical screening methods for simultaneous detection of multivitamins have gained substantial attention to ensure quality and public confidence in dietary supplements. Even so, few analytical methods have been proposed for simultaneous analysis of multivitamin constituents due to the large divergence in chemical characteristics.Objectives: In the present study, the objective was to develop a simple and rapid direct nano-electrospray ionization-tandem mass spectrometry (DI-nano-ESI-MS/MS) method for targeted detection of water soluble vitamins, fat soluble vitamins, amino acids, royal jelly, ginkgo biloba, and ginseng in a dietary supplement. The applicability of dilute-and-shoot-based DI-nano-ESI-MS/MS to analyze the same tested compounds and their related metabolites in clinical samples was also examined.
    Methods: Intact urine mixed with the ionization solvent was loaded (4-μL aliquot) into a nanospray (NS) capillary of 1-μm tip diameter. The NS capillary was then fitted into an off-line ion source at a distance of 5 mm from MS aperture. The sample was directly injected by applying a voltage of 1.1 kV, producing a numerous of m/z peaks for analysis in mere minutes.
    Results: The DI-nano-ESI-MS/MS method successfully identified almost all dietary supplement components, as well as a plethora of component-related metabolites in clinical samples. In addition, a new merit of the proposed method for the detection of index marker and chemical contaminants as well as subspecies identification was investigated for further quality evaluation of the dietary supplement.
    Conclusions: The previous findings illustrated that DI-nano-ESI-MS/MS approach can emerge as a powerful, high throughput, and promising analytical tool for screening and accurate detection of various pharmaceuticals and ingredient in dietary supplements as well as biological fluids.
    Keywords:  Amino acids; Botanical extracts; Direct nano-electrospray ionization mass spectrometry; Human urine; Multivitamins; Royal jelly
    DOI:  https://doi.org/10.1016/j.jare.2020.06.009
  16. Drug Test Anal. 2020 Nov 05.
    Lalonde K, Barber A, Ayotte C.
      Testosterone doping in sports is detected through the measurement of the carbon isotopic signature (δ13 C) of testosterone and its metabolites in urine. A critical step in achieving accurate and precise δ13 C values during compound-specific stable carbon isotope analysis (CSIA) is the removal of interfering matrix components. To this end, the World Anti-Doping Agency (WADA) recommends the use of high performance liquid chromatography (HPLC) as a method of sample pre-treatment. We provide a description of an automated two-dimensional HPLC purification (2D-HPLC) method for urine extracts that has made possible the CSIA of underivatized steroids, requiring only 36 minutes per sample. Eight urinary steroids including testosterone (T), dehydroepiandrosterone (DHEA) and 4 of their metabolites as well as two endogenous reference compounds were collected during HPLC purification. Comparative GC chromatograms are used to contrast the efficiency of 2D purification to a previously established 1D-HPLC method. The two-dimensional purification leads to improved sample purity, while simultaneously decreasing the analysis time, allowing for unprecedented sample throughput. Precision of δ13 C for all analyzed compounds in negative and positive controls was 0.5‰ or better, which is comparable to the precision of pure reference materials at similar intensities. To the authors' knowledge, this is the first description of a 2D-HPLC purification method adapted for routine doping control CSIA analysis of underivatized steroids in the literature, although the approach has recently been adopted for acetylated steroids.1.
    DOI:  https://doi.org/10.1002/dta.2964