bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒05‒03
thirty-five papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 15. pii: S1570-0232(20)30272-5. [Epub ahead of print]1146 122117
      The measurements of steroids in biological fluids are of importance for the diagnosis and treatment of many diseases. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has a high specificity and accuracy for the steroid analysis, whereas it has a lower analysis throughput, which could become a big issue in clinical practice. One of the promising solutions to this issue is the multiplexing of samples in the same injection. In this study, the utility of the sample-multiplexing by the derivatization using multiple analogous reagents was evaluated for enhancing the throughput of the LC/ESI-MS/MS assays of steroids. The plasma 17α-hydroxyprogesterone (17OHP), which is a diagnostic marker for the 21-hydroxylase deficiency, was chosen as the model analyte. The four plasma samples (20 μL each) were separately derivatized with one of four different analogous Girard-type reagents, combined, then injected together into the LC/ESI-MS/MS. By this method, four plasma samples could be analyzed within a single LC run. The developed method could significantly reduce the total LC run time (about 2/5 for 32 samples, compared with the conventional method) with a satisfactory sensitivity (lower limit of quantification 0.5 ng/mL), precision (intra- and inter-assay RSDs ≤ 4.0% and ≤ 3.5%, respectively) and accuracy (97.6-106.7%), and negligible matrix effect. The developed method had a satisfactory applicability for the quantification of 17OHP in the cord plasma samples.
    Keywords:  17α-Hydroxyprogesterone; Derivatization; Girard-type reagent; LC/ESI-MS/MS; Sample-multiplexing
  2. Anal Chem. 2020 Apr 27.
      Natural product chemistry, microbiology, and food, human, and plant metabolomics represent a few sources of complex metabolomics data generated by mass spectrometry. Among the medley of software tools used to handle these datasets, no universal tool can qualitatively, quantitatively, or statistically address major biological questions or tasks. CycloBranch 2, an open and platform-free software, at least now provides the de novo generation of molecular formulas of unknown compounds in both liquid chromatography/mass spectrometry and mass spectrometry imaging datafiles. For imaging files, this database-free approach was documented in the bimodal image fusion and characterization of three small molecules, including metallophores. The fine isotope ratio data filtering step distinguished 34S/13C2 and 41K/13C2 features. The standalone software package is implemented in C++ and can be downloaded from and used under GNU General Public License.
  3. Metabolomics. 2020 May 01. 16(5): 64
      INTRODUCTION: When analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr-Purcell-Meiboom-Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites.OBJECTIVES: Our aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples.
    METHODS: We compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares-Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated.
    RESULTS: We found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability, ~ 85% of all peaks had an adequately low coefficient of variation (< 30%) in replicate samples.
    CONCLUSION: Overall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.
    Keywords:  Classification; High-throughput; LED; Large scale; Metabolomics; NMR
  4. Curr Protoc Plant Biol. 2020 Jun;5(2): e20109
      Lipids are fascinating due to their chemical diversity, which is especially vast in the plant kingdom thanks to the high plasticity of the plant biosynthetic machinery. Lipidomic studies aim to simultaneously analyze a large number of lipid compounds of diverse classes in a given sample. The method presented here uses liquid chromatography-mass spectrometry (LC-MS)-based lipidomic profiling in a relatively fast, robust, and high-throughput manner for high-coverage quantification and annotation of lipophilic compounds. Protocols cover sample preparation, LC-MS-based measurement, and data extraction and annotation. An extensive lipid library for triacylglycerols, galactolipids, and phospholipids is provided. The extended profiling described here could be used in a range of applications and is suitable for integration with other omic datasets. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Sample preparation and metabolite extraction Basic Protocol 2: Liquid chromatography-mass spectrometry (LC-MS) analysis Basic Protocol 3: Data extraction, annotation, and quantification.
    Keywords:  fatty acids; lipid class; lipid species; lipidomics; lipids; lipophilic compounds; liquid chromatography-mass spectrometry
  5. Metabolites. 2020 Apr 28. pii: E175. [Epub ahead of print]10(5):
      Red blood cells are constantly exposed to reactive species under physiological or pathological conditions or during administration of xenobiotics. Regardless of the source, its accurate quantification is paramount in the area of theragnostics, which had been elusive up until now. Even if there are a lot of approaches to evaluate the oxidative stress, very sensitive methods are missing for the blood system. We therefore sought to apply a highly sensitive approach, by liquid chromatography coupled to mass spectrometry (UPLC-MS), for the quantification of reactive species such as superoxide radical and hydrogen peroxide using dihydroethidium (DHE) and coumarin boronic acid (CBA) probes respectively through the detection of 2-hydroxyethidium (2OH-E+) and 7-hydroxycoumarin (COH). The use of the high-resolution mass spectrometry associated to UPLC ensured a selective detection of superoxide and hydrogen peroxide in the blood system under diverse conditions such as oxidized red blood cells (RBCs), untreated and treated parasitized RBCs. Moreover, this technique allowed the determination of reactive species in human plasma. This protocol provides a huge opportunity for in-depth study of several pathological conditions vis-a-vis their treatment in modern medicine.
    Keywords:  Plasmodium falciparum; human plasma; hydrogen peroxide species; liquid-chromatography; mass spectrometry; microvesicles; red blood cells; superoxide radicals
  6. J Pharm Biomed Anal. 2020 Apr 17. pii: S0731-7085(20)30347-2. [Epub ahead of print]186 113302
      Oxidative stress is a pathological condition characterized by an imbalance between body's antioxidant defenses and oxidizing agents, resulting in damage of endogenous molecules. These products can be used as markers of oxidative conditions; in particular, isoprostanes (IsoPs) come from the reaction of arachidonic acid with reactive oxygen species (ROS) and are currently defined as gold markers of oxidative stress in urine. Our main goal was the development of a reliable analytical method for the determination and quantification of the IsoPs in human urine by dispersive Liquid-Liquid Micro Extraction (dLLME) coupled with micro Solid Phase Extraction (μSPE) clean-up and HPLC-MS/MS analysis. The selected compounds are present in very small concentration in urine, furthermore, due to relevant matrix effect, they are challenging for ESI-MS/MS analysis. This approach provided selectivity and sensitivity for 8-isoprotaglandine F2α (8-iso-PGF2α), the "gold" OS marker, together with the main isomers. dLLME extraction allowed a significant enrichment factor and μSPE clean-up provided the removal of ion-suppressing compounds from the sample resulting in low matrix effect. The chromatographic separation was also challenging as the target compounds possess very similar chemical characteristics, so experimental conditions were carefully tuned. The reported method represents a useful tool for the detection of IsoPs in urine taking advantage of the combination of dLLME extraction and μSPE clean-up; overall recoveries were above 50 % and matrix effects were ≤15 %, with LOQs ranging between 0.020 and 0.060 ng mL-1. The procedure is easy to use and rapid allowing the removal of interfering compounds and matrix effect maintaining a highly sensitive determination.
    Keywords:  Dispersive Liquid Liquid Micro Extraction; HPLC-MS/MS; Isoprostanes; Micro SPE; Urine
  7. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Apr 18. pii: S1570-0232(20)30210-5. [Epub ahead of print]1146 122122
      A rapid procedure for the determination of amphenicol antibiotics in human urine by liquid chromatography with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is proposed. The presence of thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in the human body can be attributed to their administration to treat certain diseases or by eating food of animal origin. The TAP, FF and CAP excreted in urine is mainly in the form of glucuronide conjugates, although their free forms may also be excreted to a lesser extent. In the procedure described, the enzymatic hydrolysis of amphenicol glucuronide forms in urine was carried out using β-glucuronidase and sulfatase at pH 5 (37 °C, overnight) in order to discriminate the free and conjugated forms. Then, amphenicol antibiotics were submitted to dispersive liquid-liquid microextraction (DLLME) for preconcentration. All the parameters affecting DLLME efficiency were optimized, and the following conditions were selected: 0.9 g NaCl in 10 mL of urine, to which 1.2 mL methanol (as dispersant solvent) and 1 mL of 4-methyl-2-pentanone (as extractant solvent) were added. The absence of a matrix effect allowed quantification of the samples against aqueous standards. Detection limits were 29, 6 and 3 pg mL-1 for TAP, FF and CAP, respectively. Relative standard deviations were calculated to evaluate the intra- and inter-day precision and values lower than 10% were obtained in all cases. The trueness of the method was tested through recovery studies, obtaining satisfactory values (83-104%). Ten urine samples obtained from volunteers were analysed and all of them were free of the studied antibiotics.
    Keywords:  Amphenicols; Dispersive liquid-liquid microextraction; Glucuronide; Human urine; Liquid chromatography; Quadrupole time-of-flight mass spectrometry
  8. J Pharm Biomed Anal. 2020 Apr 05. pii: S0731-7085(20)30052-2. [Epub ahead of print]186 113294
      Biogenic amines (BA) are a broad group of biologically active substances, the presence of which in the human body can provide important diagnostic information for many various pathologies, including chronic inflammation. In this work, a capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of twelve BA (histamine, serotonin, dopamine, norepinephrine, epinephrine, putrescine, cadaverine, spermine, spermidine, tyramine, tryptamine, phenylethylamine) in human urine as potential biomarkers of inflammatory bowel diseases (IBD). The electrophoretic separations were carried out in an uncoated fused silica capillary (I.D. 50 μm) using 50 mM formic acid (pH 2.0) as a background electrolyte. A reliable identification of the analytes was based on the combination of time resolution in CE and mass resolution in triple quadrupole MS/MS. The total analysis time of the proposed CEMS/MS method was less than 10 min with the limits of detection in the range of 4.47-144 ng/mL. The intra- and inter-day accuracy ranged in the intervals 89.75-109.4% and 89.99-110.2%, respectively, with the RSD values for the intra- and inter-day precision lower than 14 and 13 %, respectively. The recovery values for the samples spiked at three concentration levels ranged from 81.73-105.6% with a precision not exceeding 9.9 %. The favorable performance parameters of the CEMS/MS method highlighted its usefulness for routine clinical applications. In this work, the CEMS/MS method was applied, for the first time, to the analytical profiling of the BA in clinical human samples. The obtained results showed a statistically significant decrease of serotonin and norepinephrine, and an increase of histamine and spermidine, in the studied group of IBD patients when compared with the control group. These findings could be utilized in studying and clarifying the mechanisms of IBD or relevant therapy.
    Keywords:  Capillary electrophoresis; Catecholamines; Crohn’s disease; Histamine; Polyamines; Triple quadrupole mass spectrometry
  9. Sci Rep. 2020 Apr 27. 10(1): 7042
      A method for simultaneous determination of ten pyrethroid insecticides residues in edible mushrooms was developed. The samples were pretreated by a quick, easy, cheap, effective, rugged (QuEChERS) method. The ten pyrethroid insecticides were extracted from six kinds of edible mushrooms using acetonitrile and subsequently cleaned up by octadecylsilane (C18) or primary secondary amine (PSA). Instrumental analysis was completed in 16 min using gas chromatography-tandem mass spectrometry (GC-MS/MS). The overall average recoveries in the six kinds of edible mushrooms at three levels (10, 100 and 1000 μg kg-1) ranged from 72.8% to 103.6%. The intraday and interday relative standard deviations (RSD) were lower than 13.0%. The quantification limits were below 5.57 μg kg-1 in different matrices. The results demonstrated that the method is convenient for the quick detection of pyrethroid insecticides in edible mushrooms.
  10. Anal Chem. 2020 Apr 30.
      Diagnostic and predictable gas-phase ion-molecule reactions have emerged as a potential alternative to collision-activated dissociation in tandem mass spectrometry (MS2) experiments performed to gain structural information for unknown organic compounds, such as drug metabolites, in complex mixtures. However, the applicability of this approach for analyzing metabolites at physiologically relevant concentrations has not been determined. In this study, HPLC/MS2 experiments based on gas-phase ion-molecule reactions of protonated model compounds were successfully conducted at nanomolar and picomolar analyte concentrations. As the analyte concentration decreased, the signal-to-noise ratio of the HPLC peaks decreased more than the signal-to-noise ratio of the mass spectrometer peaks. Therefore, the HPLC part of this analysis was the primary limiting factor for each analyte (rather than the ion-molecule reactions). The ion-molecule reaction limits of detection ranged from 50 pM to 250 nM, with the average being 50-100 nM. Since all compounds had ion-molecule reaction detection limits below 500 nM, the detection limits are within the physiologically relevant range for in vivo studies of drugs and drug metabolites. When considering only mass spectrometry, the number of ion isolation events (one in MS2 experiments involving ion-molecule reactions or two in MS3 experiments involving CAD of products formed upon ion-molecule reactions) and the subsequent CAD in the MS3 experiments were the most important limiting factors. Indeed, the limit of detection for the MS3 experiments was 250 nM, about three times higher than the average ion-molecule reaction detection limit of 75 nM but still within physiologically relevant concentrations.
  11. Anal Chem. 2020 May 01.
      Top-down mass spectrometry has become the main method for intact proteoform identification, characterization, and quantitation. Because of the complexity of top-down mass spectrometry data, spectral deconvolution is an indispensable step in spectral data analysis, which groups spectral peaks into isotopic envelopes and extracts monoisotopic masses of precursor or fragment ions. The performance of spectral deconvolution methods relies heavily on their scoring functions, which distinguish correct envelopes from incorrect ones. A good scoring function increases the accuracy of deconvoluted masses reported from mass spectra. In this paper, we present EnvCNN, a convolutional neural network-based model for evaluating isotopic envelopes. We show that the model outperforms other scoring functions in distinguishing correct envelopes from incorrect ones and that it increases the number of identifications and improves the statistical significance of identifications in top-down spectral interpretation.
  12. Anal Bioanal Chem. 2020 Apr 29.
      In this study, both conventional one-dimensional liquid chromatography (1DLC) and comprehensive two-dimensional liquid chromatography (2DLC) coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF MS) were used for full-scale lipid characterization of lipid extracts from zebrafish embryos. We investigated the influence on annotated lipids and different separation mechanisms (HILIC, C18, and PFP), and their different orders arranged in the first and the second dimensions. As a result, the number of lipid species annotated by conventional one-dimensional LC-MS was between 212 and 448. In contrast, the number of individual lipids species annotated by C18×HILIC, HILIC×C18, and HILIC×PFP were 1784, 1059, and 1123, respectively. Therefore, it was evident that the performance of comprehensive 2DLC, especially the C18×HILIC method, considerably exceeded 1DLC. Interestingly, a comparison of the HILIC×C18 and C18×HILIC approaches showed, under the optimized conditions, similar orthogonality, but the effective separation power of the C18×HILIC was much higher. A comparison of the HILIC×C18 and the HILIC×PFP methods demonstrated that the HILIC×PFP separation had superior orthogonality with a small increase on its effective peak capacity, indicating that the HILIC×PFP combination maybe a promising platform for untargeted lipidomics in complex samples. Finally, from the comprehensive lipid profiling respective, the C18×HILIC was selected for further studies.
    Keywords:  Comprehensive two-dimensional liquid chromatography; Conventional one-dimensional liquid chromatography; Untargeted lipidomics; Zebrafish
  13. Anal Biochem. 2020 Apr 23. pii: S0003-2697(20)30250-5. [Epub ahead of print] 113718
      Glutamine residues susceptible to transglutaminase-catalyzed crosslinking can be identified by incorporation of dansyl cadaverine or biotin cadaverine. Bacterial transglutaminase and human transglutaminase 2 were used to modify residues in beta-casein with dansyl cadaverine. Bacterial transglutaminase was used to modify residues in human butyrylcholinesterase with biotin cadaverine. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector searches of mass spectrometry data. The MS/MS spectra from modified casein included intense peaks at 336.2, 402.2, and 447.2 for fragments of dansyl cadaverine adducts on glutamine. The MS/MS spectra from modified butyrylcholinesterase included intense peaks at 329.2, 395.2, and 440.2 for fragments of biotin cadaverine adducts on glutamine. No evidence for transglutaminase-catalyzed adducts on glutamic acid, aspartic acid, or asparagine was found. Consistent with expectation, it was concluded that bacterial transglutaminase and human transglutaminase 2 specifically modify glutamine. The characteristic ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine are useful markers for modified peptides.
    Keywords:  Protein Prospector; biotin cadaverine; butyrylcholinesterase; casein; dansyl cadaverine; mass spectrometry; transglutaminase
  14. Anal Chem. 2020 Apr 28.
      Since last decade there is a growing interest of RNA modifications analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we proposed a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. A first total digestion of purified RNA, prior CE-MS/MS analysis, enables to identify nucleoside modifications. Then, using a bottom-up approach, sequencing of RNAs and mapping of modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.
  15. J Agric Food Chem. 2020 Apr 30.
      An analytical program for multiclass, multiresidue residue analysis to qualitatively and quantitatively determine veterinary drug compounds in game meats by LC-MS/MS has been developed and validated. The method was validated for the analysis of muscle from bison, deer, elk, and rabbit to test for 112 veterinary drug residues from the following drug classes: β-agonists, anthelmintics, anti-inflammatory drugs, corticosteroids, fluoroquinolones, β-lactams, macrolides, nitroimidazoles, phenicols, polypeptides, sulfonamides, tetracyclines, thyreostats, and tranquilizers. Muscle was extracted using a simple and quick procedure based on a solvent extraction with 80% ACN/water and sample clean-up with dispersive SPE. The compounds of interest were separated using a Waters HSS T3 column and detected by tandem mass spectrometry with rapid polarity switching to detect both negatively and positively charged ions in a single run. Recoveries were calculated using extracted matrix matched calibration curves for each type of matrix. The average accuracy of fortified compounds ranged from 95.6% to 101% at the target quantitative validation level in the four matrices. The method was also validated as a qualitative screening method where all sample responses were compared to a single extracted-matrix matched calibrant at the target testing level (5 or 25 ng/g). Samples demonstrating a presumptive positive above the threshold value were re-extracted and analyzed with a five-point matrix matching extracted calibration curve. Since the beginning of this survey program, 360 samples have been analyzed for veterinary drug residues in game meats. Antibiotic or tranquilizer residues have been identified in deer (chlortetracycline, haloperidol, and tulathromycin), and rabbit (sulfadiazine).
  16. Molecules. 2020 Apr 27. pii: E2039. [Epub ahead of print]25(9):
      This study aimed to develop and validate a sensitive liquid chromatography-coupled tandem mass spectrometry method for the quantification of LDD-2614, an indirubin derivative and novel FLT3 inhibitor, in rat plasma. In addition, the developed analytical method was applied to observe the pharmacokinetic properties of LDD-2614. Chromatographic separation was achieved on a Luna omega C18 column using a mixture of water and acetonitrile, both containing 0.1% formic acid. Quantitation was performed using positive electrospray ionization in a multiple reaction monitoring (MRM) mode. The MRM transitions were optimized as m/z 426.2→113.1 for LDD-2614 and m/z 390.2→113.1 for LDD-2633 (internal standard), and the lower limit of quantification (LLOQ) for LDD-2614 was determined as 0.1 ng/mL. Including the LLOQ, the nine-point calibration curve was linear with a correlation coefficient greater than 0.9991. Inter- and intraday accuracies (RE) ranged from -3.19% to 8.72%, and the precision was within 9.02%. All validation results (accuracy, precision, matrix effect, recovery, stability, and dilution integrity) met the acceptance criteria of the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety guidelines. The proposed method was validated and demonstrated to be suitable for the quantification of LDD-2614 for pharmacokinetics studies.
    Keywords:  LC-MS/MS; LDD-2614; indirubin derivative; novel FLT3 inhibitor; pharmacokinetics
  17. Anal Chim Acta. 2020 May 22. pii: S0003-2670(20)30226-9. [Epub ahead of print]1112 34-45
      Untargeted mass spectrometry (MS) workflows are more suitable than targeted workflows for high throughput characterization of complex biological samples. However, analysis workflows for untargeted methods are inadequate for characterization of complex samples that contain multiple classes of compounds as each chemical class might require a different type of data processing approach. To increase the feasibility of analyzing MS data for multi-class/component complex mixtures (i.e., mixtures containing more than one major class of biomolecules), we developed a neural network-based approach for classification of MS data. In our in silico fractionation (iSF) approach, we utilize a neural decision tree to sequentially classify biomolecules based on their MS-detected isotopic patterns. In the presented demonstration, the neural decision tree consisted of two supervised binary classifiers to positively classify polypeptides and lipids, respectively, and a third supervised network was trained to classify lipids into the eight main sub-categories of lipids. The two binary classifiers assigned polypeptide and lipid experimental components with 100% sensitivity and 100% specificity; however, the 8-target classifier assigned lipids into their respective subclasses with 95% sensitivity and 99% specificity. Here, we discuss important relationships between class-specific chemical properties and MS isotopic envelopes that enable analyte classification. Moreover, we evaluate the performance characteristics of the utilized networks.
    Keywords:  Chemometrics; Feedforward neural network; Isotopic envelope; Mass spectrometry; Neural decision tree
  18. Adv Biosyst. 2020 Apr 30. e1900310
      Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >104 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs; and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis.
    Keywords:  extracellular vesicles; lipoproteins; liquid biopsies
  19. Am J Respir Crit Care Med. 2020 Apr 28.
    Keywords:  bronchoalveolar lavage fluid; exhaled breath condensate; gas chromatography-mass spectrometry; liquid chromatography-mass spectrometry; microbiome
  20. Food Chem. 2020 Apr 15. pii: S0308-8146(20)30678-6. [Epub ahead of print]324 126816
      Identification and quantification of triacylglycerols (TAGs) in salmon muscle tissue were conducted using electrospray ionization (ESI)-MS/MS in a triple quadrupole mass spectrometer. The confirmation of three fatty acid moieties of individual TAGs was determined using the multiple neutral loss (NL) scanning mode. A total of 98 TAGs were identified, and the predominant TAG species were 16:0-18:0-20:5 (10.4%), 18:1-18:2-22:6 (9.0%), and 18:0-18:1-22:6 (16.4%) in salmon muscle tissue. NL scanning was an effective means to confirm the three fatty acid moieties of the TAGs, leading to the rapid and accurate identification of individual TAGs. To the best of our knowledge, this is the first application of multiple neutral loss scanning to identify TAGs in salmonoid tissue, and many TAG species have been newly identified (i.e., 18:1-18:2-22:6, 16:0-18:2-20:5, 18:1-18:2-20:5, etc.). This study showed that the shotgun lipidomic approach along with NL scans is a useful means for studying TAG metabolism in fish.
    Keywords:  ESI; Fish oil; MS/MS; Neutral loss; Neutral loss scan; Salmon muscle tissue; TAGs; Triacylglycerols
  21. Nat Commun. 2020 Apr 28. 11(1): 2057
      Mass spectrometry (MS)-based targeted lipidomics enables the robust quantification of selected lipids under various biological conditions but comprehensive software tools to support such analyses are lacking. Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and analytically and prove that it is capable to generate large targeted experiments to analyze blood and to dissect lipid-signaling pathways such as in human platelets.
  22. Bioanalysis. 2020 Apr 28.
      A U(H)PLC-MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.
    Keywords:  APAP; DILI; UHPLC–MS; acetaminophen; glutathione; metabolites; plasma analysis
  23. Pract Lab Med. 2020 Aug;21 e00160
      Objectives: Vitamin B6 deficiency is associated with a wide spectrum of clinical syndromes. While vitamin B6 status is primarily assessed by measuring the biologically active form of the vitamin, pyridoxal 5-phosphate (PLP), concurrent measurement of the final metabolite 4-pyridoxic acid (PA) can provide additional information regarding supplement intake and hypophosphatasia. The aim of this study is to develop a simple method traceable to the NIST standard reference material 3950 for simultaneous detection of PLP and PA.Design & methods: A one-step reverse phase HPLC method with fluorescence detection was developed by evaluating different derivatization conditions, the use of an internal standard and different calibration strategies. The assay analytical performance was evaluated.
    Results: Pre-column derivatization with semicarbazide showed the best overall performance in terms of signal to noise ratio, retention time and peak shape when compared to pre- or post-column derivatization with chlorite, pre-column or in-mobile phase derivatization using sodium bisulfite. The final method provided an analytical measurement range from 7.8 to 350 ​nmol/L for PLP and 3.3-339 ​nmol/L for PA, total imprecision <15% and <5% for PLP and PA respectively. Calibration against the NIST standard produced measured values within 3% of NIST assigned PLP values. The use of 4-deoxypyridoxine as internal standard did not improve precision or accuracy when compared to calibration using 5-level external standards.
    Conclusions: This method combines derivatization and protein precipitation in one step and is traceable to NIST standard reference material 3950. It is simple and reliable for routine evaluation of vitamin B6 nutrition status.
    Keywords:  4-Pyridoxic acid; Liquid chromatography; NIST traceable; Pyridoxal 5-phosphate; Vitamin B6
  24. Electrophoresis. 2020 Apr 26.
      Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4'- and C1-monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min, in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative- and positive-ionization modes. The uniqueness of the NACE-ESI-MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4') and acylation pattern of native lipid A species or those designed for therapeutic applications. This article is protected by copyright. All rights reserved.
    Keywords:  acylation isomerism; lipid A; nonaqueous capillary electrophoresis-tandem mass spectrometry; normal and reverse polarity; phosphorylation isomerism
  25. Anal Chem. 2020 Apr 30.
      DNA cytosine modifications are important epigenetic marks. To elucidate their roles by a large scale of comparative studies, it is important to quantify the abundance of DNA cytosine modifications accurately. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a golden option. The performance of LC-MS/MS is heavily dependent on the ionization or protonation of target analytes. Initially, we found that two factors, DNA hydrolysate buffer and residual co-eluted nucleosides, might greatly suppress the protonation of 5-(hydroxymethyl)-2'-deoxycytidine (5hmdC). Surprisingly, ammonium bicarbonate could eliminate the suppression caused by both factors. Mechanistically, ammonium bicarbonate increases the protonation capacity in gas phase and facilitates proton transfer to the target nucleosides. Benefiting from these findings, we developed a suppression-free, sensitive, and robust ultra-high-performance LC-MS/MS assay for massive detection of three DNA cytosine modifications, including 5-methyl-2'-deoxycytidine (5mdC), 5hmdC, and 5-formyl-2'-deoxycytidine (5fdC). By 30 consecutive analyses, the relative standard deviation (RSD) of the 5hmdC and 5fdC peak areas was 2.0 % and 3.2 %, respectively. In this case, no stable isotope-labeled standard was required for internal calibration. We further performed a comprehensive profiling of DNA cytosine modifications in 26 tissues of age-different C57BL/6N mice. Interestingly, we found that, only did liver 5hmdC abundance increase with increasing age of adult mice, suggesting that liver 5hmdC might be a potential indicator of age in adulthood.
  26. Bioanalysis. 2020 Apr 28.
      Background: The continual need for the development and validation of ultra-sensitive (low pg/ml) LC-MS/MS assays in the pharmaceutical industry is largely driven by the ultra-low analyte exposure or very low sample volume. Methodology: Strategies and systematic approaches for sensitivity enhancement are provided which cover all aspects of a LC-MS/MS bioanalysis. A case study where such strategies were applied for the validation of a 5.0 pg/ml assay for a STING agonist is discussed. Conclusion: Analytical protocols were developed to extract analytes from large volume of plasma samples (600 and 400 μl) with high throughput. The guidance provided in this publication can serve as a resource to influence LC-MS/MS method development activities.
    Keywords:  LC–MS/MS; LLQ ; carryover; nonspecific binding; pharmacokinetic; sensitivity enhancement
  27. J Pharm Biomed Anal. 2020 Apr 06. pii: S0731-7085(19)32959-0. [Epub ahead of print]186 113291
      Silk sericin (SS) is, together with silk fibroin (SF), one of the two proteins forming the silkworm cocoon. SS is ideal ingredient for cosmetic applications in the formulation of specific products for skin care and hair due to its peculiar physical-chemical composition. SS also showed a great potential in different pharmacological and biotechnological applications, as anticancer drug, anticoagulant, cell culture additive, wound healing agent and drug delivery carrier. Reasons for SS use in biomedical applications derive from its physical-chemical composition. As a consequence, a detailed characterization of SS in terms of average molecular weight, molecular weight distribution and hydro/lipophilic character is crucial to properly address and assess its quality, cosmetic or pharmacological use. In this study, the application of different and complementary chromatographic modes allows a detailed investigation of SS protein isolated from wastewater using two diverse extraction methods. Hydrophilic interaction liquid chromatography (HILIC using an AdvanceBio Glycan Map column) and reverse phase (RP using Symmetry300 C18 column) were applied to intact protein characterization to derive data on protein hydrophilicity and on hydrophobic components of the two SS preparations (SS#1 and SS#2). A higher hydrophilic character of SS#1 was observed by HILIC trace, coherently with the preparation method used, while no significant differences in hydrophobicity were detectable in the RPLC separations. Size distribution was also defined by using a SEC-UV-MS method (using TSKgel SuperSW2000 column) properly optimized to maximize both the size selectivity and the method sensitivity. Taken together, the chromatographic data allowed to better characterize the SS samples obtained by different extraction methods, and the structural properties were correlated to their biological activities.
  28. Enferm Infecc Microbiol Clin. 2020 Apr 25. pii: S0213-005X(20)30163-4. [Epub ahead of print]
      MALDI-TOF mass spectrometry has become a reference method for the routine identification of bacterial isolates in clinical microbiology laboratories around the world. Its high specificity, user-friendliness and cost-efficiency, together with its ability to provide reliable results in less than 5min have favoured its implementation and further development. The amount of microbial species that can be identified by MALDI-TOF routinely has increased in the last few years and now it is possible to reliably identify non-tuberculous mycobacteria or closely-related species of Nocardia spp. Yeasts, both belonging to Candida and non-Candida genera can also be identified by MALDI-TOF, as well as filamentous fungi. In the latter case, both sample preparation methods and the available databases have been important factors in achieving accurate identifications at the species level. The expertise acquired over time has allowed researchers to identify microorganisms directly from clinical samples, facilitating improved management of infected patients. This expertise has also been applied to the development of a MALDI-TOF-based methodology for the detection of different antimicrobial resistance mechanisms. Therefore, future applications such as bacterial strain typing, or the detection of virulence markers seems feasible to perform with this technology. Furthermore, other emerging mass spectrometry and spectroscopy technologies may assist MALDI-TOF in the near future to carry out important tasks that nowadays are performed by time-consuming and labour-intensive methods.
    Keywords:  Antibiotic resistance mechanisms; Espectrometría de masas MALDI-TOF; Identificación; Identification; MALDI-TOF mass spectrometry; Marcadores de virulencia; Mecanismos de resistencia antibiótica; Tipado; Typing; Virulence markers
  29. Electrophoresis. 2020 May 02.
      The hyphenation of capillary electrophoresis with high-resolution mass spectrometry such as Orbitrap MS is of broad interest for the unambiguous and exceptionally sensitive identification of compounds. However, the coupling of these techniques requires a robust ionization interface that does not influence the stability of the separation voltage while coping with oxidation of the emitter tip at large ionization voltages. Herein, we present the design of a sheath-flow CE-ESI-MS interface which combines a robust and easy to operate set-up with high-resolution Orbitrap MS detection. The sheath liquid interface is equipped with a gold coated electrospray emitter which increases the stability and overall lifetime of the system. For the characterization of the interface, the spray stability and durability were investigated in dependence of the sheath-flow rate, electrospray voltage and additional gold coating. The optimized conditions were applied to a separation of angiotensin II and neurotensin resulting in limits of detection of 2.4 and 3.5 ng/mL. This article is protected by copyright. All rights reserved.
    Keywords:  CE-MS; electrospray ionization; peptide separation
  30. J Pharm Biomed Anal. 2020 Apr 17. pii: S0731-7085(20)30267-3. [Epub ahead of print]186 113301
      A novel method was developed for the determination of aucubin in rat serum by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with supramolecular solvent (SUPRAS)-based dispersive liquid-liquid microextraction (DLLME). The SUPRAS was prepared with pentanol as extraction solvent and tetrahydrofuran as dispersing agent. Based on single factor test and response surface methodology, critical parameters were optimized as: 1 mL of pentanol as extraction solvent, 4 mL of tetrahydrofuran as dispersing agent, 200 μL of SUPRAS, and vortex duration of 2.5 min. The established method was validated in terms of selectivity, linearity, accuracy, precision, recovery, stability, and applied to a pharmacokinetic study on type 1 diabetes model rats intraperitoneally administered with aucubin. The experimental results showed that the maximum concentration in serum (Cmax) and area under the serum concentration versus time curve (AUC) of aucubin in type 1 diabetic rats were higher than those in normal rats. Such pharmacokinetic alterations might presumably result from the pathological state of type 1 diabetes. The current study may provide scientific evidence and inspiring insights for clinical application of aucubin for the treatment of diabetes.
    Keywords:  Aucubin; Diabetes; Dispersive liquid-liquid microextraction; Pharmacokinetics; Supramolecular solvent; Tandem mass spectrometry; Ultra-performance liquid chromatography
  31. J Chromatogr A. 2020 Apr 09. pii: S0021-9673(20)30298-3. [Epub ahead of print] 461077
      A previous paper of this series of study put forward a basic model of an automated system for predicting detection limits and showed its application to a simple example of isocratic high-performance liquid chromatography (HPLC). This paper describes an expansion of the basic system into gradient HPLC. The most serious problem with the expansion is a long-term variation in backgrounds, called gradient baseline drifts, which in theory cannot be covered by a noise model (stationary random process) of the original system. This paper demonstrates that the above problem can be solved with modifying a parametrization procedure of the noise model. The essential role of the system is to predict the standard deviation (SD) of measurements at low concentrations from a chromatogram without repeated measurements of real samples. Laboratory-made software enables the automated assessment of the limits of detection and quantitation for each of chromatographically separated signals in a single run. Simulated background noise which consists of the stationary noise model with linear slopes is used to confirm the accuracy and reproducibility of the automated prediction. A gradient HPLC determination for cefaclor is taken as an example. The parametrization modification improves the correlation coefficient, r2, between the observed and theoretical distributions of the area measurements from 0.373 to 0.966. The statistical confidence levels of the theoretically predicted relative SDs for cefaclor were verified by comparing them with those obtained by repeated experiments (r2 = 0.989). The limits of detection (= 3.3 × SD = 18.0 µg/L) and quantitation (= 10 × SD = 54.7 µg/L) for cefaclor have signal-to-noise ratios close to the commonly adopted values, 3 and 10, respectively.
    Keywords:  Baseline drift; Detection limit; FUMI theory; Gradient elution; Quantitation limit
  32. Phytochem Anal. 2020 Apr 26.
      INTRODUCTION: In situ analysis of volatile organic compounds (VOCs) emitted by plants is an important challenge in chemical ecology. The traditional approach usually consists in trapping compounds using dynamic headspace extraction (DHS) in-field, followed by gas chromatography analysis coupled with mass spectrometry (GC-MS and/or GC-FID) in the laboratory.OBJECTIVES: In this study, we evaluated the use of the new portable Torion T-9 GC-MS system for rapid and in situ analysis of VOCs emitted by fine lavender and lavandin species.
    MATERIAL AND METHODS: All field analyses were performed using a person-portable low-thermal mass GC system coupled with a miniature toroidal ion trap mass analyser (ppGC-ITMS): Torion T-9 portable GC-MS. Subsequently, multivariate statistical analyses were performed to determine chemical differences between species.
    RESULTS: Thirty compounds were separated and detected in all lavender above-ground samples in only 3 min of analysis.
    CONCLUSIONS: The portable GC-MS device enabled a rapid in-field distinction of Lavandula species based on their detected volatile profiles.
    Keywords:  chemical ecology; lavender; portable gas chromatography; volatile organic compounds
  33. Biomed Chromatogr. 2020 May 01. e4872
      Xuefu Zhuyu Decoction (XFZYD) is a traditional Chinese medicine prescription used for the clinical treatment of traumatic brain injury (TBI). The purpose of this work was to develop a sensitive and rapid UHPLC-MS/MS method to simultaneously study the pharmacokinetics of nimodipine and eight components of XFZYD, namely, amygdalin, hydroxysafflor yellow A, rutin, liquiritin, narirutin, naringin, neohesperidin and saikosaponin A, in rats with and without TBI. MRM was highly selective in the detection of 9 analytes and the internal standard without obvious interference. The calibration curves displayed good linearity (r > 0.99) over a wide concentration range. The mean absolute recoveries of the nine analytes were 85-106%, and all matrix effects were in the range of 80-120%. The intra- and inter-day precision and accuracy were acceptable (RSD, below 15%; RE%, ±20%). The validated method was successfully applied to compare the pharmacokinetics in four experimental groups, including control rats orally administered XFZYD and TBI model rats orally administered XFZYD, XFZYD and nimodipine, or nimodipine. The results showed that herb-drug interactions occurred between XFZYD and nimodipine in the treatment of TBI, nimodipine affected the pharmacokinetics of XFZYD, and XFZYD affected the absorption, distribution and excretion of nimodipine in vivo.
    Keywords:  UHPLC-MS/MS; Xuefu Zhuyu Decoction; nimodipine; pharmacokinetics; rat plasma; traumatic brain injury
  34. Phytochem Anal. 2020 Apr 26.
      OBJECTIVE: We sought to develop a sensitive and accurate analytical method for the detection and quantification of IDP and DMADP as well as their monophosphate derivatives in crude plant extracts.METHODS: A liquid chromatography method coupled to tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) was established to measure the amounts of IDP and DMADP down to low picogram levels, which was linear over at least three orders of magnitude. Extracts were enriched using an anion exchanger, and chromatographic separation was achieved using a β-cyclodextrin column. A S-thiolodiphosphate analog of DMADP was employed as an internal standard.
    RESULTS: Dilution series of authentic compounds were used to determine the limits of detection and quantification for IDP, DMADP and their corresponding monophosphates. A survey of plant species producing varying amounts of isoprenoids showed a corresponding variation in IDP and DMADP with the ratio of DMADP/IDP ranging from 4:1 to 2:1. Trace levels of isopentenyl monophosphate (IP) and dimethylallyl monophosphate (DMAP) were also detected.
    CONCLUSION: The LC-MS/MS method described enables absolute quantification of in planta levels of IDP and DMADP for the first time. The method is also suitable for analysing bacterial and animal samples as well as enzyme assays.
    Keywords:  4-hydroxy-3-methylbut-2-enyl diphosphate reductase; dimethylallyl diphosphate; isopentenyl diphosphate; isopentenyl diphosphate isomerase; isoprenoids; methyl-D-erythritol 4-phosphate pathway; terpenes