bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒03‒29
twenty-nine papers selected by
Sofia Costa
Cold Spring Harbor Laboratory


  1. Rapid Commun Mass Spectrom. 2020 Mar 24. e8792
    Radchenko T, Kochansky CJ, Cancilla M, Wrona MD, Mortishire-Smith RJ, Kirk J, Murray G, Fontaine F, Zamora I.
      RATIONALE: Liquid chromatography/mass spectrometry is an essential tool for efficient and reliable quantitative and qualitative analysis and underpins much of contemporary drug metabolism and pharmacokinetics. Data-independent acquisition methods such as MSE have reduced potential to miss metabolites, but do not formally generate quadrupole-resolved product ion spectra. The addition of ion mobility separation to these approaches, for example, in High Definition MSE (HDMSE ) has the potential to reduce the time needed to set up an experiment and maximize the chance all metabolites present can be resolved and characterized. We compared High Definition Data-Dependent Acquisition (HD-DDA), MSE and HDMSE approaches using automated software processing with MassMetaSite and WebMetabase.METHODS: Metabolite identification was performed on incubations of glucagon-like peptide-1 (7-37) (GLP-1) and verapamil hydrochloride. The HD-DDA, MSE and HDMSE experiments were conducted on a Waters ACQUITY UPLC I-Class LC system with VION IMS QTof mass spectrometer operating under UNIFI control. All acquired data were processed using MassMetaSite able to read data from UNIFI 1.9.4. WebMetabase was used to review the detected chromatographic peaks and the spectral data interpretations.
    RESULTS: A comparison of outcomes obtained for MSE and HDMSE data demonstrated that the same structures were proposed for metabolites of both verapamil and GLP-1. The ratio of structurally matched to mismatched product ions found by MassMetaSite was slightly greater for HDMSE than for MSE , and HD-DDA, thus improving confidence in the structures proposed through the addition of ion mobility-based data acquisitions. CONCLUSIONS: HDMSE data acquisition is an effective approach for the elucidation of metabolite structures for both small molecules and peptides, with excellent accuracy and quality, requiring minimal tailoring for the compound under investigation.
    DOI:  https://doi.org/10.1002/rcm.8792
  2. Anal Chem. 2020 Mar 23.
    Ding J, Kind T, Zhu QF, Wang Y, Yan JW, Fiehn O, Feng YQ.
      Fatty acid esters of hydroxy fatty acids (FAHFAs) are a family of recently discovered lipids with important physiological functions in mammals and plants. However, low detection sensitivity in negative ionization mode mass spectrometry makes low abundant FAHFA challenging to analyze. A 2-dimethylaminoethylamine (DMED) based chemical derivatization strategy was recently reported to improve the MS sensitivity of FAHFAs by labeling FAHFAs with a positively ionizable tertiary amine group. To facilitate a reliable, high throughput and automatic annotation of these compounds, a DMED-FAHFA in-silico library containing 4,290 high-resolution tandem mass spectra covering 264 different FAHFA classes was developed. The construction of the library was based on the heuristic information from MS/MS fragmentation patterns of DMED-FAHFA authentic standards, and then applied the patterns to computer-generated DMED-FAHFAs. The developed DMED-FAHFA in-silico library was demonstrated to be compatible with library search software NIST MS Search and the LC-MS/MS data processing tool MS-DIAL to guarantee high throughput and automatic annotations. Applying the in-silico library in Arabidopsis thaliana samples for profiling FAHFAs by high resolution LC-MS/MS enabled the annotation of 19 DMED-FAHFAs from 16 families, including 4 novel compounds. Using the in-silico library largely decreased the false positive annotation rate in comparison to low resolution LC-MS/MS. The developed library, MS/MS spectra and development templates are freely available for commercial and non-commercial use at https://zenodo.org/record/3606905.
    DOI:  https://doi.org/10.1021/acs.analchem.0c00172
  3. Clin Biochem. 2020 Mar 19. pii: S0009-9120(19)31378-5. [Epub ahead of print]
    Bergmann ML, Schmedes A.
      The catecholamines, epinephrine (E) and norepinephrine (NE) are important both as neurotransmitters and hormones, and measurement of E and NE in plasma is therefore of great interest in medical research. However, the low concentrations of E and NE in plasma require an analysis method that is both specific and sensitive. Plasma sample E and NE were extracted using solid phase extraction, and analyzed by an in-sample ion-pairing chromatography (IPC) LC-MS/MS method, that enables the ion-pairing reagent to be diverted to waste without entering the ion source of the mass spectrometer. The method was validated with good performance characteristics and the limit of quantification (LOQ) was found to be 0.20 and 0.02 nmol/L for NE and E, respectively. In conclusion, this work presents development and validation of a method for determining E and NE in plasma with a measuring range covering the entire reference interval for human plasma.
    Keywords:  Catecholamine; Epinephrine; In-sample Ion-pairing chromatography; Mass spectrometry; Norepinephrine; Plasma
    DOI:  https://doi.org/10.1016/j.clinbiochem.2020.03.006
  4. Anal Chim Acta. 2020 Apr 22. pii: S0003-2670(20)30199-9. [Epub ahead of print]1107 74-84
    Zhou GS, Yuan YC, Yin Y, Tang YP, Xu RJ, Liu Y, Chen PD, Yin L, Duan JA.
      A sensitive, rapid, precise and specific analytical method of hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole linear ion-trap tandem mass spectrometry (HILIC-UHPLC-QTRAP®/MS2) combined with a high-efficiency and easy sample preparation technology of ultrasound-assisted ionic liquid dispersive liquid-liquid microextraction (UA-IL-DLLME) was developed to investigate neurotransmitters (NTs) in mild cognitive impairment, mild dementia and moderate dementia patients' urine samples. Firstly, the UA-IL-DLLME parameters were optimized using Plackett-Burman screening and rotatable central composite design, and the main optimal conditions were obtained: ultrasound power of 307 W, ultrasound time of 4.3 min and agitation time of 4.8 min. Secondly, HILIC-UHPLC-QTRAP®/MS2 method was developed to simultaneously determine 15 underivatized NTs in urine samples. The analysis results of clinical samples showed that some NTs such as γ-aminobutyric acid (GABA), acetylcholine (Ach) and glutamic acid (Glu) presented significant differences in different dementia stages. Finally, multivariate analysis based on the combination of principal component analysis and supervised counter propagation artificial neural network was developed for comprehensive analysis of the obtained clinical data sets. As a result, GABA and Glu were simultaneously presented meaningful contribution for classification of samples, and might be considered as potential differential compounds to the urine samples from cluster patients with different dementia stages. In summary, the presented strategy of preparation, analysis and statistics might be used to investigate NTs in different clinical biological fluids.
    Keywords:  Dementia; Hydrophilic interaction chromatography; Ionic liquid dispersive liquid–liquid microextraction; Neurotransmitters; Ultrasound
    DOI:  https://doi.org/10.1016/j.aca.2020.02.027
  5. Methods Mol Biol. 2020 ;2138 207-216
    Jayasena T, Bustamante S, Clement J, Welschinger R, Caplan GA, Sachdev PS, Braidy N.
      Nicotinamide adenine dinucleotide (NAD+) and its related metabolites (NADome) are important endogenous analytes that are thought to play important roles in cellular metabolism, inflammation, oxidative stress, cancer, neurodegeneration, and aging in mammals. However, these analytes are unstable during the collection of biological fluids, which is a major limiting factor for their quantitation. Herein, we describe a highly robust and quantitative method using liquid chromatography coupled to tandem mass spectrometry to quantify the NADome in whole blood, plasma, mononuclear cells, platelets, cerebrospinal fluid (CSF), and urine. This methodology represents a "gold standard" of measure for understanding biological pathways and developing targeted pharmacological interventions to modulate NAD+ biosynthesis and NAD-dependent mediators in health and disease.
    Keywords:  Aging; Cancer; Inflammation; Metabolism; NAD+; NADome; Neurodegeneration; Oxidative stress
    DOI:  https://doi.org/10.1007/978-1-0716-0471-7_13
  6. Bioanalysis. 2020 Mar 25.
    Schwieler L, Trepci A, Krzyzanowski S, Hermansson S, Granqvist M, Piehl F, Venckunas T, Brazaitis M, Kamandulis S, Lindqvist D, Jones AD, Erhardt S, Brundin L.
      Aim: Kynurenine metabolites are potential modulators of psychiatric disease. We aimed to develop a highly sensitive biochemical analysis of cerebrospinal fluid (CSF) tryptophan (TRP) metabolites, to investigate the stability of metabolites and to confirm our previous findings of aberrant CSF quinolinic acid (QUIN) and picolinic acid (PIC) in suicide attempters using this method. Methodology & results: Ten CSF TRP metabolites were analyzed with ultraperformance LC-MS/MS. The method showed small intra- and interassay variation. Metabolites were stable following freeze-thaw cycles. A decreased CSF PIC/QUIN ratio was found in suicide attempters. Conclusion: The feasibility of reliably determining CSF TRP metabolites were demonstrated, including separation of the two isomers PIC and nicotinic acid (NA) and the finding of a reduced PIC/QUIN ratio replicated in suicide attempters.
    Keywords:  LC–MS/MS; nicotinic acid; picolinic acid; quinolinic acid; suicide; tryptophan-kynurenine pathway
    DOI:  https://doi.org/10.4155/bio-2019-0303
  7. Anal Chem. 2020 Mar 26.
    Heuillet M, Millard P, Y Cissé M, K Linares L, Letisse F, Manie SN, Le Cam L, Portais JC, Bellvert F.
      Studies of the topology, functioning and regulation of metabolic systems are based on two main types of information which can be measured by mass spectrometry: the (absolute or relative) concentration of metabolites and their isotope incorporation in 13C-labeling experiments. These data are currently obtained from two independent experiments because the 13C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the 13C-mass fractions from which its 13C-isotopologue distribution (CID) is quantified. Here, we developed a generic method with a dedicated processing workflow to obtain these two information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of two both the number of cultivations to perform and the number of samples to collect, prepare and analyze. The proposed approach is based on an IS labeled with other isotope(s) which can be resolved from the 13C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the PERK kinase, a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino-acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.
    DOI:  https://doi.org/10.1021/acs.analchem.9b05709
  8. J Proteome Res. 2020 Mar 27.
    Pinto FG, Mahmud I, Harmon TA, Rubio VY, Garrett TJ.
      Spectrometric methods with rapid biomarker detection capacity through untargeted metabolomics are becoming essential in the clinical cancer research. Liquid chromatography-mass spectrometry (LC-MS) is a rapidly developing metabolomic-based biomarker technique due its high sensitivity, reproducibility, and separation efficiency. However, its transla-tion to clinical diagnostics is often limited due to long data acquisition times (~20 min/sample) and laborious sample extraction procedures when employed for large-scale metabolomics studies. Here, we developed a segmented flow approach coupled with high-resolution mass spectrometry (SF-HRMS) for untargeted metabolomics which has the capability to acquire data in less than 1.5 min/sample with robustness and reproducibility relative to LC-HRMS. The SF-HRMS results demonstrate the capability for screening metabolite-based urinary biomarkers associated with prostate cancer (PCa). The study shows that SF-HRMS-based global metabolomics has the potential to evolve into a rapid biomarker screening tool for clinical research.
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00006
  9. Anal Chem. 2020 Mar 26.
    McLean C, Kujawinski EB.
      Untargeted metabolomics experiments provide a snapshot of cellular metabolism, but remain challenging to interpret due to the computational complexity involved in data processing and analysis. Prior to any interpretation, raw data must be processed to remove noise and to align mass-spectral peaks across samples. This step requires selection of dataset-specific parameters, as erroneous parameters can result in noise inflation. While several algorithms exist to automate parameter selection, each depends on gradient descent optimization functions. In contrast, our new parameter optimization algorithm, AutoTuner, obtains parameter estimates from raw data in a single step as opposed to many iterations. Here, we tested the accuracy and the run time of AutoTuner in comparison to isotopologue parameter optimization (IPO), the most commonly-used parameter selection tool, and compared the resulting parameters' influence on the quality of feature tables after processing. We performed a Monte Carlo experiment to test the robustness of AutoTuner parameter selection, and found that AutoTuner generated similar parameter estimates from random subsets of samples. We conclude that AutoTuner is a desirable alternative to existing tools, because it is scalable, highly robust, and very fast (~100-1000X speed improvement from other algorithms going from days to minutes). AutoTuner is freely available as an R package through BioConductor.
    DOI:  https://doi.org/10.1021/acs.analchem.9b04804
  10. Methods Protoc. 2020 Mar 24. pii: E23. [Epub ahead of print]3(1):
    Notardonato I, Passarella S, Ianiri G, Di Fiore C, Russo MV, Avino P.
      In this paper, an analytical protocol was developed for the simultaneous determination of phthalates (di-methyl phthalate DMP, di-ethyl phthalate DEP, di-isobutyl phthalate DiBP, di-n-butyl phthalate DBP, bis-(2-ethylhexyl) phthalate DEHP, di-n-octyl phthalate DNOP) and bisphenol A (BPA). The extraction technique used was the ultrasound vortex assisted dispersive liquid-liquid microextraction (UVA-DLLME). The method involves analyte extraction using 75 µL of benzene and subsequent analysis by gas chromatography combined with ion trap mass spectrometry (GC-IT/MS). The method is sensitive, reliable, and reproducible with a limit of detection (LOD) below 13 ng g-1 and limit of quantification (LOQ) below 22 ng g-1 and the intra- and inter-day errors below 7.2 and 9.3, respectively. The method developed and validated was applied to six honey samples (i.e., four single-use commercial ones and two home-made ones. Some phthalates were found in the samples at concentrations below the specific migration limits (SMLs). Furthermore, the commercial samples were subjected to two different thermal stresses (24 h and 48 h at 40 °C) for evidence of the release of plastic from the containers. An increase in the phthalate concentrations was observed, especially during the first phase of the shock, but the levels were still within the limits of the regulations.
    Keywords:  DLLME; GC-IT/MS; analytical protocol; bisphenol A; dispersive solvent; honey; honeycomb; phthalates; thermal stress; ultrasound
    DOI:  https://doi.org/10.3390/mps3010023
  11. Metabolomics. 2020 Mar 25. 16(4): 44
    Poupin N, Vinson F, Moreau A, Batut A, Chazalviel M, Colsch B, Fouillen L, Guez S, Khoury S, Dalloux-Chioccioli J, Tournadre A, Le Faouder P, Pouyet C, Van Delft P, Viars F, Bertrand-Michel J, Jourdan F.
      INTRODUCTION: To interpret metabolomic and lipidomic profiles, it is necessary to identify the metabolic reactions that connect the measured molecules. This can be achieved by putting them in the context of genome-scale metabolic network reconstructions. However, mapping experimentally measured molecules onto metabolic networks is challenging due to differences in identifiers and level of annotation between data and metabolic networks, especially for lipids.OBJECTIVES: To help linking lipids from lipidomics datasets with lipids in metabolic networks, we developed a new matching method based on the ChEBI ontology. The implementation is freely available as a python library and in MetExplore webserver.
    METHODS: Our matching method is more flexible than an exact identifier-based correspondence since it allows establishing a link between molecules even if a different level of precision is provided in the dataset and in the metabolic network. For instance, it can associate a generic class of lipids present in the network with the molecular species detailed in the lipidomics dataset. This mapping is based on the computation of a distance between molecules in ChEBI ontology.
    RESULTS: We applied our method to a chemical library (968 lipids) and an experimental dataset (32 modulated lipids) and showed that using ontology-based mapping improves and facilitates the link with genome scale metabolic networks. Beyond network mapping, the results provide ways for improvements in terms of network curation and lipidomics data annotation.
    CONCLUSION: This new method being generic, it can be applied to any metabolomics data and therefore improve our comprehension of metabolic modulations.
    Keywords:  Lipidomics; Mapping; Metabolic networks; Ontology
    DOI:  https://doi.org/10.1007/s11306-020-01663-5
  12. Metabolites. 2020 Mar 24. pii: E122. [Epub ahead of print]10(3):
    Mitchell JM, Flight RM, Moseley HNB.
      Despite instrument and algorithmic improvements, the untargeted and accurate assignment of metabolites remains an unsolved problem in metabolomics. New assignment methods such as our SMIRFE algorithm can assign elemental molecular formulas to observed spectral features in a highly untargeted manner without orthogonal information from tandem MS or chromatography. However, for many lipidomics applications, it is necessary to know at least the lipid category or class that is associated with a detected spectral feature to derive a biochemical interpretation. Our goal is to develop a method for robustly classifying elemental molecular formula assignments into lipid categories for an application to SMIRFE-generated assignments. Using a Random Forest machine learning approach, we developed a method that can predict lipid category and class from SMIRFE non-adducted molecular formula assignments. Our methods achieve high average predictive accuracy (>90%) and precision (>83%) across all eight of the lipid categories in the LIPIDMAPS database. Classification performance was evaluated using sets of theoretical, data-derived, and artifactual molecular formulas. Our methods enable the lipid classification of non-adducted molecular formula assignments generated by SMIRFE without orthogonal information, facilitating the biochemical interpretation of untargeted lipidomics experiments. This lipid classification appears insufficient for validating single-spectrum assignments, but could be useful in cross-spectrum assignment validation.
    Keywords:  Random Forest; SMIRFE; lipid category; lipidomics; machine learning; metabolomics
    DOI:  https://doi.org/10.3390/metabo10030122
  13. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 19. pii: S1570-0232(20)30136-7. [Epub ahead of print]1143 122069
    Luiz Oenning A, Birk L, Eller S, Franco de Oliveira T, Merib J, Carasek E.
      In this study, the use of switchable hydrophilicity solvent with a simple and low-cost lab-made device for the extraction procedure in homogeneous liquid-liquid microextraction is proposed for the first time in the determination of antidepressants in human urine. The antidepressants studied consisted of fluoxetine, amitriptyline, nortriptyline, imipramine, desipramine and sertraline. The optimization of the main parameters that can influence on the extraction efficiency was performed through multivariate approaches. The analytes were separated and identified by gas chromatography coupled to mass spectrometry (GC-MS). The optimal extraction conditions consisted of using N,N-dimethylcyclohexylamine (DMCHA) as the switchable hydrophilicity solvent (SHS), 500 µL of urine sample previously diluted with ultrapure water at 1:1 ratio (v/v), 200 μL of a mixture of SHS:HCl 6 mol L-1 (1:1 v/v), 600 μL of NaOH 10 mol L-1 and 3 min of extraction time. A volume of 40 µL of diphenylamine at concentration of 500 µg L-1 (20 ng) was used as internal standard. The method developed was in-house validated, providing coefficients of determination higher than 0.995 for all analytes, limits of detection (LOD) from 0.02 to 0.88 µg L-1, limits of quantification (LOQ) from 0.05 to 2.92 µg L-1, relative recoveries of 68 to 102%, intra-day precision from 0.5 to 15.9%, inter-day precision from 4.2 to 19.3%, selectivity and robustness. The method proposed was successfully applied in five human urine samples from a Toxicological Information Center located in Porto Alegre (Brazil). The results demonstrated that the µP-SHS-HLLME approach is highly cost-effective, rapid, simple and environmentally-friendly with satisfactory analytical performance.
    Keywords:  Antidepressants; GC-MS; Homogeneous liquid-liquid microextraction; Switchable hydrophilicity solvents; Urine
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122069
  14. J Vis Exp. 2020 Mar 08.
    Mohammad K, Jiang H, Hossain MI, Titorenko VI.
      Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/µL. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.
    DOI:  https://doi.org/10.3791/60616
  15. Anal Chim Acta. 2020 Apr 22. pii: S0003-2670(20)30191-4. [Epub ahead of print]1107 92-100
    Wang T, Chen X, Luan C, Wu J.
      Cell heterogeneity of tumor tissues is one of the causes of cancer recurrence after chemotherapy. Cell subtype identification in tumor tissues of specific cancer is critical for precision medicine and prognosis. As the structural and functional components of cells, lipids are closely related to the apparent morphology of cells. They are potential biomarkers of species of cancers and can be used to classify different cancer cell types, but it remains a challenge to establish a stable cell differentiation model and extend it to tumor tissue cell subtype differentiation. Here we describe a lipid profiling method based on nanostructure assisted laser desorption/ionization mass spectrometry (NALDI-MS), which could classify five hepatocellular carcinoma (HCC) cell lines and discriminate subtype of mixed cells and tumor tissues. The NALDI target was patterned with array of sample spots containing vertical silicon nanowires (Si NWs). Owing to its high ability to absorb laser energy, the vertical Si NWs can help to generate abundant lipid ions of cell extracts without need of organic matrix. Combined with statistical analysis methods, twenty-two ion peaks distributed in four MS peak clusters were selected as potential biomarkers to distinguish the subtype of the five HCC cell lines. Peak normalization was performed within each MS peak cluster to reduce the variation of peak intensity in batch to batch analysis. Compared to full-spectrum normalization method, the inner-cluster normalization method could help to distinguish cell subtype more stably and accurately. The molecular structure of these biomarkers was identified and sorted into two classes including phosphatidylcholine (PE, PI, PG, PA, PS) and glycosphingolipid (LacCer, ST). Furthermore, the established method was successfully applied to identify the major HCC cell subtype in mixed cell samples and xenograft tumor tissues as well as drug response test, showing great potential in precision medicine and prognosis.
    Keywords:  Cell and tissue subtype; Drug susceptibility; High throughput silicon nanowires array chip; Lipidomics; Matrix-free laser desorption/ionization
    DOI:  https://doi.org/10.1016/j.aca.2020.02.019
  16. Anal Chem. 2020 Mar 26.
    Graton J, Hernández-Mesa M, Normand S, Dervilly G, Le Questel JY, Le Bizec B.
      A wide range of Collision Cross Section (CCS) databases for different families of compounds have recently been established from Ion Mobility Mass Spectrometry (IM-MS) measurements. Nevertheless, the need to validate these new data sets in order to provide the necessary confidence about the use of this parameter is increasingly expressed by the scientific community. If such a validation requires that complementary mass spectrometry experiments are conducted, it also appears that alternative strategies can contribute to the validation of such empirical data. In particular, in silico approaches are relevant to compute theoretical CCS values, to be compared with experimental ones. A recently published CCS database for 300 steroids allowed observing experimentally significant deviations of the expected CCS vs. m/z correlations for some compounds. The present work attempts to rationalize such deviations with Density Functional Theory (DFT) calculations. MN15/6-311++G(d,p) investigations have been carried out, starting with a conformational analysis of a sample of 20 selected steroids and the determination of their preferred gas-phase ionization site. CCS values were then computed and compared to the experimental data. This approach allowed rationalizing the experimental trends, providing an accurate description of key properties of the various steroids considered.
    DOI:  https://doi.org/10.1021/acs.analchem.0c00357
  17. Talanta. 2020 Jun 01. pii: S0039-9140(20)30138-7. [Epub ahead of print]213 120847
    Bambauer TP, Wagmann L, Maurer HH, Weber AA, Meyer MR.
      The analytical proof of a toxic mushroom and/or plant ingestion at an early stage of a suspected intoxication can be crucial for fast therapeutic decision making. Therefore, comprehensive analytical procedures need to be available. This study aimed to develop a strategy for the qualitative analysis of α- and β-amanitin, psilocin, bufotenine, muscarine, muscimol, ibotenic acid, and ricinine in human urine by means of hydrophilic interaction liquid chromatography-high resolution MS/MS (HILIC-HRMS/MS). Urine samples were prepared by hydrophilic-phase liquid-liquid extraction using dichloromethane and subsequent solid-phase extraction and precipitation, performed in parallel. Separation and identification of the biomarkers were achieved by HILIC using acetonitrile and methanol as main eluents and Orbitrap-based mass spectrometry, respectively. The method was validated as recommended for qualitative procedures and tests for selectivity, carryover, and extraction recoveries were included to also estimate the robustness and reproducibility of the sample preparation. Limits of identification were 1 ng/mL for α- and β-amanitin, 5 ng/mL for psilocin, bufotenine, muscarine, and ricinine, and 1500 ng/mL and 2000 ng/mL for ibotenic acid and muscimol, respectively. Using γ-amanitin, l-tryptophan-d5, and psilocin-d10 as internal standards, compensation for variations of matrix effects was shown to be acceptable for most of the toxins. In eight urine samples obtained from intoxicated individuals, α- and β-amanitin, psilocin, psilocin-O-glucuronide, muscimol, ibotenic acid, and muscarine could be identified. Moreover, psilocin-O-glucuronide and bufotenine-O-glucuronide were found to be suitable additional targets. The analytical strategy developed was thus well suited for analyzing several biomarkers of toxic mushrooms and plants in human urine to support therapeutic decision making in a clinical toxicology setting. To our knowledge, the presented method is by far the most comprehensive approach for identification of the included biomarkers in a human matrix.
    Keywords:  Biomarker; HILIC-HRMS/MS; Mushroom intoxication; Mushroom toxins; Ricinine
    DOI:  https://doi.org/10.1016/j.talanta.2020.120847
  18. Toxicol Mech Methods. 2020 Mar 25. 1-26
    Adomshick V, Pu Y, Veiga-Lopez A.
      Adipogenic differentiation is the process by which preadipocytes become mature adipocytes, cells that store energy and regulate metabolic homeostasis. During differentiation, neutral lipids that accumulate in adipocytes can be detected using stains and used as an index of cell differentiation. However, imaging tools for evaluating intracellular lipid droplets remain at their infancy. Nutrition, stress, or chemical exposure can dysregulate adipogenic differentiation and lipid metabolism. Therefore, the aims of this study were to develop an accurate, standardized approach to quantify lipid droplet size of mature adipocytes and a clustering approach to analyze the total lipid content per adipocyte. For the lipid droplet analysis, we used two approaches, the free online computer software of reference, ImageJ, and another free online computer software, CellProfiler. For ImageJ, we used an already developed macro designed to identify particles and quantify their area, and for CellProfiler, we developed a new analysis pipeline. Our results show that CellProfiler is able to accurately identify a greater number of lipid droplets compared to ImageJ. A clustering analysis is also possible using CellProfiler which allows for the quantification of total lipid content per individual adipocyte to provide insight into single-cell responsiveness to adipogenic stimuli. CellProfiler streamlines the lipid droplet phenotypic analysis of adipocytes compared to more traditional analysis methods. In conclusion, this novel image analysis tool can provide with a more precise evaluation of lipid droplet and adipogenesis dysregulation, a critical need in the understanding of metabolic disorders.
    Keywords:  adipocyte; image segmentation; lipid droplet; quantitative analyses
    DOI:  https://doi.org/10.1080/15376516.2020.1747124
  19. Chem Res Toxicol. 2020 Mar 24.
    Ousji O, Ohlund L, Sleno L.
      Bisphenol A (BPA) metabolism has been investigated using several in vitro models, including human and rat liver microsomes and subcellular (S9) fractions, as well as human-recombinant Cytochrome P450 3A4 (CYP3A4) expressed in Supersomes, for a comprehensive look at all possible metabolic pathways. By an untargeted approach using liquid chromatography coupled to a high-resolution quadrupole-time of flight mass spectrometer, we were able to detect a large number of known Phase I and Phase II metabolites of BPA, as well as several previously uncharacterized ones. A detailed fragmentation study of BPA and its detected metabolites was crucial to confirm structures. Isotope-labeled BPA analogs were highly useful for the structural elucidation of many metabolites. These results contribute to a better understanding of BPA metabolism, including pathways that may introduce additional toxicity, as well as help with the assessment of BPA exposure in different biological matrices.
    DOI:  https://doi.org/10.1021/acs.chemrestox.0c00042
  20. J Pharm Biomed Anal. 2020 Mar 09. pii: S0731-7085(19)33083-3. [Epub ahead of print]185 113245
    Roseboom IC, Thijssen B, Rosing H, Mbui J, Beijnen JH, Dorlo TPC.
      A highly sensitive method was developed to quantitate the antileishmanial agent paromomycin in human plasma, with a lower limit of quantification of 5 ng/mL. Separation was achieved using an isocratic ion-pair ultra-high performance liquid chromatographic (UPLC) method with a minimal concentration of heptafluorobutyric acid, which was coupled through an electrospray ionization interface to a triple quadrupole - linear ion trap mass spectrometer for detection. The method was validated over a linear calibration range of 5 to 1000 ng/mL (r2≥0.997) with inter-assay accuracies and precisions within the internationally accepted criteria. Volumes of 50 μL of human K2EDTA plasma were processed by using a simple protein precipitation method with 40 μL 20 % trichloroacetic acid. A good performance was shown in terms of recovery (100 %), matrix effect (C.V. ≤ 12.0 %) and carry-over (≤17.5 % of the lower limit of quantitation). Paromomycin spiked to human plasma samples was stable for at least 24 h at room temperature, 6 h at 35 °C, and 104 days at -20 °C. Paromomycin adsorbs to glass containers at low concentrations, and therefore acidic conditions were used throughout the assay, in combination with polypropylene tubes and autosampler vials. The assay was successfully applied in a pharmacokinetic study in visceral leishmaniasis patients from Eastern Africa.
    Keywords:  Assay; Bioanalytical validation; Human plasma; Ion-pair liquid chromatography; Paromomycin; Tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.jpba.2020.113245
  21. J Chromatogr A. 2020 Mar 13. pii: S0021-9673(20)30257-0. [Epub ahead of print] 461045
    Farhadpour M, Maghari S, Rezadoost H, Bagheri M, Ghassempour A.
      New zwitterionic (ZIC) stationary phases (SPs) are synthesized with the click and conventional bonding of tyrosine to silica gel. Infrared spectra and elemental analysis demonstrate the successful click and conventional bonding of this ZIC group on silica particles by the surface coverage including 2.36 and 0.75 µm m-2, respectively. Given the above-mentioned explanation, the present study evaluated the retention mechanism and chromatographic manners of polar compounds on these new materials under hydrophilic interaction liquid chromatography (HILIC) conditions. Based on the results, the Click-Tyrosine Stationary Phase provided good HILIC characteristics when it was applied to separate phenolic compounds, amino acids, alkaloids, and nucleobases compared to bare silica gel SP and even conventional tyrosine SPs. Further, this new Click-Tyrosine-SP represented appropriate HILIC features and column efficiency (the theoretical plate number was up to 50,000 plates m-1 for thebaine). Furthermore, the study investigated the effect of solute polarity (the number of the hydroxyl group of phenolic compounds) and hydrophobicity (the number of the side chain of aliphatic amino acids) on retention behaviors. Finally, some important factors were studied as the potential variables for guiding the retention behavior of the polar compound in HILIC condition including solvent composition, salt concentration, and the buffer pH of the mobile phase.
    Keywords:  Click-tyrosine-sp; HILIC; Hydrophilic interaction liquid chromatography; Zwitterion
    DOI:  https://doi.org/10.1016/j.chroma.2020.461045
  22. Curr Protoc Bioinformatics. 2020 Jun;70(1): e98
    Blimkie T, Lee AH, Hancock REW.
      MetaBridge is a web-based tool designed to facilitate the integration of metabolomics with other "omics" data types such as transcriptomics and proteomics. It uses data from the MetaCyc metabolic pathway database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) to map metabolite compounds to directly interacting upstream or downstream enzymes in enzymatic reactions and metabolic pathways. The resulting list of enzymes can then be integrated with transcriptomics or proteomics data via protein-protein interaction networks to perform integrative multi-omics analyses. MetaBridge was developed to be intuitive and easy to use, requiring little to no prior computational experience. The protocols described here detail all steps involved in the use of MetaBridge, from preparing input data and performing metabolite mapping to utilizing the results to build a protein-protein interaction network. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Mapping metabolite data using MetaCyc identifiers Basic Protocol 2: Mapping metabolite data using KEGG identifiers Support Protocol 1: Converting compound names to HMDB IDs Support Protocol 2: Submitting mapped genes produced by MetaBridge for protein-protein interaction (PPI) network construction.
    Keywords:  metabolite mapping; metabolomics; multi-omics integration
    DOI:  https://doi.org/10.1002/cpbi.98
  23. Bioanalysis. 2020 Mar 25.
    Leaney AE, Horner C, Grace PB, Mawson DH.
      Aim: A surrogate matrix is needed to quantify 25-hydroxyvitamin D3 in dried whole blood (DWB). To date, there has been limited guidance on approaches for quantification of endogenous analytes in atypical matrices, such as DWB. Methods: Different surrogate matrices were investigated in a systematic process using an LC-MS/MS assay. Performance assessment was made using quality controls of DWB with different hematocrits. Results & conclusion: Suitability of both phosphate-buffered saline containing bovine serum albumin and washed red blood cells recombined with phosphate-buffered saline containing bovine serum albumin as a surrogate matrix was demonstrated across a range of concentrations and hematocrits representative of expected endogenous analyte samples.
    Keywords:  25-hydroxyvitamin D3; VAMS; dried whole blood; endogenous; hematocrit; quantification; surrogate; whole blood
    DOI:  https://doi.org/10.4155/bio-2020-0012
  24. Biotechnol Bioeng. 2020 Mar 26.
    Xue P, Si T, Mishra S, Zhang L, Choe K, Sweedler JV, Zhao H.
      Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs), key components of crucial nutrients, soaps, industrial chemicals, and fuels. However, our ability to control the specificity of microbially synthesized FFAs is still limited for medium-chain fatty acids (MCFAs), which is mainly due to lack of high-throughput approaches for FFA analysis. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. Specially, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and the m/z peak ratio at 730 and 758 was used as an index for improved MCFA production. This yeast colony-based method can be performed at a rate of ~2 second per sample, which is much faster than the gold standard gas chromatography (GC)-MS (typically > 30 min per sample). To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified 9 mutants that resulted in improved MCFA production relative to the wild-type strain. This colony-based MALDI-ToF MS screening method provides an alternative approach for engineering microbial fatty acid compositions in a high-throughput manner. This article is protected by copyright. All rights reserved.
    Keywords:  Directed evolution; Free fatty acids; High-throughput screening; Mass spectrometry; Metabolic engineering
    DOI:  https://doi.org/10.1002/bit.27343
  25. J Pharm Biomed Anal. 2020 Mar 12. pii: S0731-7085(20)30017-0. [Epub ahead of print]185 113250
    Rigo-Bonnin R, Tiraboschi JM, Álvarez-Álvarez M, Pérez-Fernández GA, Sanjuás-Iglesias M, Scévola S, Niubó J, Videla S, Podzamczer D.
      Bictegravir is a novel integrase strand transfer inhibitor, administrated in co-formulation with tenofovir alafenamide and emtricitabine (Biktarvy®), indicated in the management of HIV-1 infection in patients not previously treated with antiretroviral therapy. Bictegravir is highly bound to plasma proteins, and this significantly determines its clearance, solubility, and activity. These characteristics are crucial determinants of bictegravir penetration into human body compartments, as the central nervous system. We developed and validated UHPLC-MS/MS procedures to measure total and unbound bictegravir concentrations in plasma and cerebrospinal fluid. Simple protein precipitation with acetonitrile was implemented to prepare plasma and cerebrospinal fluid samples. Sample preparation was preceded by ultrafiltration for measuring unbound bictegravir concentrations. Chromatographic separations were achieved on an Acquity® UHPLC® BEHTM (2.1 × 100 mm id, 1.7 μm) reverse-phase C18 column using an isocratic mobile phase 20:80 (v/v) water/acetonitrile with 0.1% formic. Bictegravir and its internal standard (bictegravir-15N d2) were detected by electrospray ionization mass spectrometry in positive and multiple reaction monitoring modes, using transitions of 450.2→289.2/145.4 and 453.2→289.2, respectively. Ultrafiltration procedures presented non-specific bindings of (8.6 ± 1.2) % for bictegravir in plasma and (26.6 ± 3.1) % for bictegravir in cerebrospinal fluid. Linearity was observed between (10.70-8560) μg/L, (1.07-856.0) μg/L for total and unbound bictegravir in plasma, and 0.107-26.75 μg/L for total and unbound bictegravir in cerebrospinal fluid. Imprecisions, absolute relative biases, normalized-matrix factors, and normalized-recoveries were ≤14.4%, ≤13.8%, (97.4-102.5) %, and (99.8-105.1) %, respectively. No significant interferences and carry-over were observed. The validated UHPLC-MS/MS procedures could be useful for pharmacokinetic and pharmacodynamic studies.
    Keywords:  Cerebrospinal fluid; UHPLC-MS/MS; plasma; total bictegravir; ultrafiltration; unbound bictegravir
    DOI:  https://doi.org/10.1016/j.jpba.2020.113250
  26. J Chromatogr A. 2020 Mar 12. pii: S0021-9673(20)30244-2. [Epub ahead of print] 461041
    Behpour M, Nojavan S, Asadi S, Shokri A.
      The current study presents for the first time a combination of the gel electromembrane extraction (GEL-EME) and switchable hydrophilicity solvent-based homogeneous liquid-liquid microextraction (SHS-HLLME) methods which can be used as an efficient hyphenated extraction procedure. This coupled method, which was followed by GC-FID, was applied for quantification of antidepressants (desipramine, clozapine, and citalopram) in biological and wastewater samples. The effective parameters of both GEL-EME and SHS-HLLME procedures were optimized. Using an agarose gel membrane, analytes were extracted from 7.0 mL of the sample solution to 500 µL of the aqueous acceptor solution. The maximum extraction of analytes of interest was obtained under the optimized conditions (pH of acceptor solution, 5.0; pH of gel membrane, 5.0; pH of sample solution, 7.0, voltage value, 30 V; and extraction time, 30 min). Then, the acceptor solution was transferred to the extraction cell and the SHS-HLLME procedure was conducted again under the optimized conditions. Dipropylamine (50 µL) was selected as an extraction solvent. The introduced technique exhibited good linearities with coefficients of determinatin (R2) higher than 0.983 and an acceptable linear range of 5.0-1000 ng/mL. Accordingly, the limit of detection was ≤ 1.0 ng/mL (S/N = 3) for all analytes, and the high enrichment factors were obtained in the range of 178.7-194.8. Moreover, the corresponding repeatability was from 4.0 to 8.7% (n = 3). The proposed method was successfully utilized to determine trace levels of the drugs in human serum, wastewater, and breast milk samples.
    Keywords:  Breast milk; Gel electromembrane extraction; Homogeneous liquid-liquid microextraction; Human serum; Switchable solvent; Wastewater
    DOI:  https://doi.org/10.1016/j.chroma.2020.461041
  27. Talanta. 2020 Jun 01. pii: S0039-9140(20)30098-9. [Epub ahead of print]213 120807
    Ali AAF, Danielson ND.
      A C18 column was modified with the anionic amino acid surfactant lauroyl-l-glutamate (LLG) to facilitate the separation of ten short-chain aliphatic carboxylic acids (oxalic, tartaric, malic, malonic, lactic, acetic, maleic, citric, fumaric, and succinic). The developed method was proven to be fast, versatile, and environmentally friendly. After the coating of the column using 1% LLG solution and optimizing chromatographic conditions such as pH and temperature, near baseline resolution of the ten carboxylic acids within 4 min with excellent peak shape at pH = 1.8 using 100% H2O acidified with sulfuric acid was possible. Although the design of this stationary phase, with the hydrophilic group at the end of the alkyl chain, seems to be in contrast to such columns designed for a totally aqueous mobile phase that have a polar (often amide) group embedded near the silica surface, no evidence of phase collapse was noted. Linear relationships of ln retention factor (k) versus 1/Temperature (T) (van't Hoff plots) were generated for all the acids indicating a single retention mechanism was likely. As the pH of the mobile phase decreased, the analyte retention factors increased due to the increase of the fraction of the analyte with neutral charge (alpha zero). The surfactant amide linkage, being electron donating, increased the pKa of the more acidic carboxyl group of glutamic acid so both carboxyl groups were protonated (neutral) at pH 1.8. The exact nature of the retention mechanism is uncertain but there certainly seems to be a pronounced hydrophobic component due to the large difference in retention of fumaric acid and methyl fumarate at pH 1.8. In addition, eleven beverage samples were analyzed for their aliphatic carboxylic acid contents. The results showed that malic, fumaric, and citric acids were the most common carboxylic acids in natural beverages with concentrations as high as 6432 ppm of malic acid in organic apple juice, 64 ppm of fumaric acid in organic concord juice, and 6543 ppm citric acid in strawberry lemonade juice.
    Keywords:  Aliphatic polar carboxylic acids; Amino acid surfactant; Green liquid chromatography
    DOI:  https://doi.org/10.1016/j.talanta.2020.120807
  28. Anal Chim Acta. 2020 Apr 22. pii: S0003-2670(20)30194-X. [Epub ahead of print]1107 101-106
    Wang CH, Su H, Chou JH, Lin JY, Huang MZ, Lee CW, Shiea J.
      Multiple solid phase microextraction (mSPME) combined with thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) was developed to rapidly characterize trace analytes in aqueous solution. A number of commercial available SPME fibers (from 2 to 10 fibers) were simultaneously used for extracting the analytes in solution. The fibers were then bundled together on a holder and subjected for the ambient mass spectrometric analysis. Good linearity for calibration (R2 = 0.9995) and low limit of quantification (<1 ppb) were achieved by using 10 SPME fibers coated with polyacrylate (PA) to extract bisphenol A. It was also found that the analyte signals increased with the number of SPME fibers for extraction. Uncontroversial, a shorter extraction time was required by using mSPME to reach the same level of analyte signal as that by using single SPME fiber for a longer extraction time. Trace bisphenol A (4-20 ppb) in the polycarbonate (PC) baby milk bottles was rapidly detected using mSPME-TD-ESI/MS and the analysis was completed within 1 min. The use of multiple SPME fibers coated with different materials enable the concentration of different type of analytes in the solution. Ibuprofen, bisphenol A (BPA), and 4-n-nonylphenol (4-n-NP) were simultaneously detected by using PA and polydimethylsiloxane (PDMS) coated fibers for extraction.
    Keywords:  Ambient mass spectrometry; Solid phase microextraction; Thermal desorption-electrospray ionization
    DOI:  https://doi.org/10.1016/j.aca.2020.02.022