bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒04‒25
twenty-two papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Front Chem. 2021 ;9 619770
      The cannabis market is expanding exponentially in the United States. As state-wide legalization increases, so do demands for analytical testing methodologies. One of the main tests conducted on cannabis products is the analysis for terpenes. This research focused on implementation of accelerated solvent extraction (ASE), utilizing surrogate matrix matching, and evaluation of traditional vs. more modern sample introduction techniques for analyzing terpenes via gas chromatography-mass spectrometry (GC-MS). Introduction techniques included Headspace-Syringe (HS-Syringe), HS-Solid Phase Microextraction Arrow (HS-SPME Arrow), Direct Immersion-SPME Arrow (DI-SPME Arrow), and Liquid Injection-Syringe (LI-Syringe). The LI-Syringe approach was deemed the most straightforward and robust method with terpene working ranges of 0.04-5.12 μg/mL; r 2 values of 0.988-0.996 (0.993 average); limit of quantitation values of 0.017-0.129 μg/mL (0.047 average); analytical precisions of 2.58-9.64% RSD (1.56 average); overall ASE-LI-Syringe-GC-MS method precisions of 1.73-14.6% RSD (4.97 average); and % recoveries of 84.6-98.9% (90.2 average) for the 23 terpenes of interest. Sample workflows and results are discussed, with an evaluation of the advantages/limitations of each approach and opportunities for future work.
    Keywords:  accelerated solvent extraction (ASE); gas chromatography–mass spectrometry (GC-MS); solid-phase microextraction (SPME); solid-phase microextraction Arrow (SPME Arrow); terpenes
  2. Ther Drug Monit. 2021 Apr 13.
      BACKGROUND: Routine therapeutic drug monitoring is a promising approach for the rational use of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase (ALK) inhibitors. The purpose of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of five EGFR-TKIs (afatinib, dacomitinib, erlotinib, gefitinib, and osimertinib) and three ALK inhibitors (alectinib, ceritinib, and crizotinib).METHODS: A 100-microliter aliquot of serum was diluted with 100 μL of 1% aqueous ammonia containing internal standards and then purified using the supported liquid extraction method. LC-MS/MS was conducted in positive ionization mode, and the method was validated according to published guidelines.
    RESULTS: Calibration curves were linear across concentration ranges examined. The intra- and inter-assay accuracies were 90.7-110.7% and 94.7-107.6%, respectively. All intra- and inter-assay imprecision values were ≤10.1%. The EGFR-TKIs and ALK inhibitors examined in this study, except osimertinib, which could be stored on ice for at least 5 h, were stable at room temperature for 3 h. For the internal standard-normalized matrix factors, the mean recovery and percent coefficient of variation values ranged between 54-112% and 1.7-11.7%, respectively. This method successfully determined serum concentrations of afatinib, alectinib, erlotinib, gefitinib, and osimertinib in clinical samples. Serum levels of kinase inhibitors consistently reflected those reported in previous studies.
    CONCLUSIONS: An LC-MS/MS method suitable for the simultaneous determination of five EGFR-TKIs and three ALK inhibitors in serum was developed and validated. The newly developed method enabled the determination of five of eight target drugs examined in clinical samples. However, a large number of clinical samples need to be analyzed to verify the usefulness of the method.
  3. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Apr 01. pii: S1570-0232(21)00165-3. [Epub ahead of print]1173 122685
      The potential therapeutic effects of various phytocannabinoids and the availability of multiple cannabis-based medicines make it desirable to have an analytical method that simultaneously quantifies a wide range of cannabinoids in blood, beyond delta-9-tetrahydrocannabinol and its metabolites. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of 18 phytocannabinoids and cannabinoid metabolites in serum was developed and validated. The method enables simultaneous detection of delta-9-tetrahydrocannabinol, cannabidiol, cannabinol, cannabigerol, cannabichromene, cannabicyclol, tetrahydrocannabivarin and cannabidivarin and their acidic precursors tetrahydocannabinolic acid A, cannabidiolic acid, cannabinolic acid, cannabigerolic acid, cannabichromenic acid, cannabicyclolic acid, tetrahydrocannabivarinic acid and cannabidivarinic acid as well as the delta-9-tetrahydrocannabinol metabolites 11-hydroxy-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol. Limits of detection ranged from 0.0004 to 1 ng/mL and limits of quantification ranged from 0.004 to 2 ng/mL. Calibration curves of all analytes were linear over the whole calibration range. Recovery rates of 52 to 86% were obtained for all analytes except for cannabicyclol (49%), 11-nor-9-carboxy-tetrahydrocannabinol (46%), cannabichromenic acid (44%) and cannabidivarinic acid (36%). Acceptable bias and precision data were demonstrated for all analytes. The method was successfully applied to 55 forensic serum samples, obtained from the Institute of Legal Medicine Mainz.
    Keywords:  Cannabinoids; LC-MS/MS; Method validation; Serum level; Solid phase extraction
  4. J Biosci Bioeng. 2021 Apr 17. pii: S1389-1723(21)00059-1. [Epub ahead of print]
      The ability to reduce sample volume required for gas chromatography-mass spectrometry (GC/MS) metabolome analyses is limited by the effects of blank matrices. In this study, a GC/MS metabolome analytical method requiring only 5 μL of plasma obtained by fingertip puncture, was developed, while minimizing the adverse effects of blank matrices. The applicability of the newly developed method was investigated using mice tail venous blood. Removing the effects of blank matrices greatly affected the detection repeatability for trace amounts. The newly developed method has higher metabolite coverage and higher sensitivity than those of the conventional method. This study is the first to demonstrate that comparable, or improved, metabolome profile data can be obtained with one-tenth the plasma volume required for the conventional method.
    Keywords:  Blank matrix; Derivatization; Gas chromatography–mass spectrometry; Metabolomics; Micro-scale sample; Plasticizer
  5. Anal Chem. 2021 Apr 23.
      Metabolic networks and their dysfunction in the brain are closely associated with central nervous function and many psychogenic diseases. Thus, it is of utmost importance to develop a high-throughput imaging method for metabolic network mapping. Here, we developed a metabolic network mapping method to discover the metabolic contexts and alterations with spatially resolved information from the microregion of the brain by ambient-air flow-assisted desorption electrospray ionization mass spectrometry imaging and metabolomics analysis, which can be performed without any chemical derivatization, labels, or complex sample pretreatment. This method can map hundreds of different polar functional metabolites involved in multiple metabolic pathways, including not only neurotransmitters but also purines, organic acids, polyamines, cholines, and carbohydrates, in the rat brain. These high-coverage metabolite profile and microregional distribution information constitute complex networks that regulate advanced functions in the central nervous system. Moreover, this methodology was further used to discover not only the dysregulated metabolites but also the brain microregions involved in the pathology of a scopolamine-treated Alzheimer's model. Furthermore, this methodology was demonstrated to be a powerful visualizing tool that could offer novel insight into the metabolic events and provide spatial information about these events in central nervous system diseases.
  6. J Agric Food Chem. 2021 Apr 23.
      In addition to their important role in fat digestion, bile acids are increasingly being used as markers for various diseases. The large diversity of bile acids results from the conversion of primary and conjugated bile acids into secondary bile acids by deconjugation and dehydroxylation reactions mediated by the intestinal microbiota. Here, we describe a fast and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for absolute quantitation of 45 bile acids in human or mouse feces in combination with a simple workup and extraction procedure. Method validation outlined excellent limits of detection and quantitation, linearity, selectivity, recovery, extraction loss, and precision. To investigate the connection between microbiome alterations and bile acid metabolism, the method was applied on a Crohn's disease study including patients with histologically documented active disease or remission as well as on a model using humanized mice. As the complex mechanism including genetic and environmental factors leading to the development of Crohn's disease is so far not completely understood, the study investigates the microbial metabolism of bile acids and the potential use of bile acid profiles to predict disease state.
    Keywords:  Crohn’s disease; UHPLC-ESI-LC-MS/MS; bile acids; method validation; targeted metabolite profiling
  7. J Chromatogr A. 2021 Mar 31. pii: S0021-9673(21)00253-3. [Epub ahead of print]1645 462129
      Comprehensive two-dimensional liquid chromatography is a well-established method for the unraveling of very complex real-world samples. With regard to food and natural products such a technique turned out to be a very promising approach due to its high resolving power and improved identification capability, especially in combination with mass spectrometry. In this context, polyphenols comprise a particular complex class of bioactive compounds, due to their nature and content in commonly consumed foodstuffs, making their analysis challenging. The present contribution shows an overview of the two commonly employed approaches used for polyphenol analysis, viz. RP-LC × RP-LC and HILIC × RP-LC. Furthermore, the latest implementations as well as limitations and future perspectives are critically reported.
    Keywords:  Comprehensive two-dimensional liquid chromatography; Food analysis; HILIC × RP-LC; Natural products; Polyphenols; RP-LC × RP-LC
  8. Anal Chem. 2021 Apr 21.
      Polar phosphorylated metabolites are involved in a variety of biological processes and play vital roles in energetic metabolism, cofactor regeneration, and nucleic acid synthesis. However, it is often challenging to interrogate polar phosphorylated metabolites and compounds from biological samples. Liquid chromatography-mass spectrometry (LC/MS) now plays a central role in metabolomic studies. However, LC/MS-based approaches have been hampered by the issues of the low ionization efficiencies, low in vivo concentrations, and less chemical stability of polar phosphorylated metabolites. In this work, we synthesized paired reagents of light and heavy isotopomers, 2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (DMPI) and d3-(2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (d3-DMPI). The paired reagents of DMPI and d3-DMPI carry diazo groups that can efficiently and selectively react with the phosphate group on polar phosphorylated metabolites under mild conditions. As a proof of concept, we found that the transfer of the indole heterocycle group from DMPI/d3-DMPI to ribonucleotides led to the significant increase of ionization efficiencies of ribonucleotides during LC/MS analysis. The detection sensitivities of these ribonucleotides increased by 25-1137-fold upon DMPI tagging with the limits of detection (LODs) being between 7 and 150 amol. With the developed method, we achieved the determination of all the 12 ribonucleotides from a single mammalian cell and from a single stamen of Arabidopsis thaliana. The method provides a valuable tool to investigate the dynamic changes of polar phosphorylated metabolites in a single cell under particular conditions.
  9. Pract Lab Med. 2021 May;25 e00213
      Objectives: Despite reports highlighting citrate association with different diseases, serum citrate is scarcely used for diagnosis. Existing methods to quantify citrate are limited by their complexity and practicality of implementation. A simple and rapid NMR-based method to measure circulating citrate is described here, and its analytical performance evaluated.Design: and Methods: Citrate was quantified from NMR spectra using a non-negative linear least squares deconvolution algorithm. The analytical characteristics of the assay were evaluated using CLSI guidelines. To determine if the assay has adequate sensitivity to measure clinically relevant concentrations of citrate, the assay was used to quantify citrate in apparently healthy adults (n ​= ​553), and in the general population (n ​= ​133,576).
    Results: The LOQ for the assay was determined to be 1.48 ​mg/dL. Linearity was demonstrated over a wide range of concentrations (1.40-4.46 ​mg/dL). Coefficients of variation (%CV) for intra- and inter-assay precision ranged from 5.8-9.3 and 5.2-9.6%, respectively. Substances tested did not elicit interference with assay results. Specimen type comparison revealed <1% bias between serum and plasma samples, except for heparin plasma (3% bias). Stability was demonstrated up to 8 days at room temperature and longer at lower temperatures. In a cohort of apparently healthy adults, the reference interval was <1.48-2.97 ​mg/dL. Slightly higher values were observed in the general population.
    Conclusions: The newly developed NMR-based assay exhibits analytical characteristics that allow the accurate quantification of clinically relevant citrate concentrations. The assay provides a simple and fast means to analyze samples for research and clinical studies.
    Keywords:  1D, one dimensional; 1H, proton; CLSI, Clinical and Laboratory Standards Institute; CV, coefficient of variation; Citrate; LOB, limit of blank; LOD, limit of detection; LOQ, limit of quantitation; MS, Mass Spectrometry; Mortality; NAFLD, non-alcoholic fatty liver disease; NMR, Nuclear magnetic resonance spectroscopy; Nuclear magnetic resonance spectroscopy
  10. J Chromatogr A. 2021 Mar 22. pii: S0021-9673(21)00210-7. [Epub ahead of print]1646 462086
      Stand-alone electrospray ionization mass spectrometry (ESI-MS) has been advancing through enhancements in throughput, selectivity and sensitivity of mass spectrometers. Unlike traditional MS techniques which usually require extensive offline sample preparation and chromatographic separation, many sample preparation techniques are now directly coupled with stand-alone MS to enable outstanding throughput for bioanalysis. In this review, we summarize the different sample clean-up and/or analyte enrichment strategies that can be directly coupled with ESI-MS and nano-ESI-MS for the analysis of biological fluids. The overview covers the hyphenation of different sample preparation techniques including solid phase extraction (SPE), solid phase micro-extraction (SPME), slug flow micro-extraction/nano-extraction (SFME/SFNE), liquid extraction surface analysis (LESA), extraction electrospray, extraction using digital microfluidics (DMF), and electrokinetic extraction (EkE) with ESI-MS and nano-ESI-MS.
    Keywords:  Biological fluids; Electrospray ionization-mass spectrometry (ESI-MS); Nano-ESI-MS; Sample preparation; Stand-alone mass spectrometry
  11. Anal Chem. 2021 Apr 23.
      Proton-transfer-reaction (PTR) mass spectrometry (MS) is capable of detecting trace-level volatile organic compounds (VOCs) in gaseous samples in real time. Therefore, PTR-MS has become a popular method in many different study areas. Most of the currently reported PTR-MS applications are designed to determine volatile compounds. However, the method might be applicable for nonvolatile organic compound detection. Supercritical fluid chromatography (SFC) has been studied in the last 5 decades. This approach has high separation efficiency and predictable retention behavior, making separation optimization easy. Atmospheric ionization techniques, such as atmospheric chemical ionization (APCI) and electrospray ionization (ESI), are the most studied SFC-MS interfaces. These processes require the addition of makeup solvents to prevent precipitation or crystallization of the solute while depressurizing the mobile phase. In contrast, the PTR process is carried out in a vacuum; supercritical carbon dioxide may release solute into the PTR flow tube without a phase transition as long as it is maintained above a critical temperature. Therefore, this might constitute yet another use for the SFC-MS interface. Caffeine and a few other nonpolar compounds in supercritical carbon dioxide were successfully detected with time-of-flight MS without adding solvent by using preliminarily assembled supercritical flow injection and supercritical fluid extraction (SFE)-PTR interfaces.
  12. J Mass Spectrom. 2021 Mar 10. 56(5): e4718
      The use of ion mobility separations (IMSs) in metabolomics approaches has started to be deeply explored in the last years. In this work, the use of liquid chromatography (LC) coupled to IMS-quadrupole time-of-flight mass spectrometry (QTOF MS) has been evaluated in a metabolomics experiments using single injection of the samples. IMS has allowed obtaining cleaner fragmentation spectra, of nearly tandem MS quality, in data-independent acquisition mode. This is much useful in this research area as a second injection, generally applied in LC-QTOF MS workflows to obtain tandem mass spectra, is not necessary, saving time and evading possible compound degradation. As a case study, the smoke produced after combustion of herbal blends used to spray synthetic cannabinoids has been selected as study matrix. The smoke components were trapped in carbon cartridges, desorbed and analyzed by LC-IMS-QTOF MS using different separation mechanisms (reversed phase and HILIC) and acquiring in both positive and negative mode to widen the chemical domain. Partial Least Squares-Discriminant Analysis highlighted several compounds, and ratio between N-Isopropyl-3-(isoquinolinyl)-2-propen-1-amine and quinoline allowed differentiating between tobacco and herbal products. These two compounds were tentatively identified using the cleaner fragmentation spectra from a single injection in the IMS-QTOF MS, with additional confidence obtained by retention time (Rt) and collisional cross section (CCS) prediction using artificial neural networks. Data from this work show that LC-IMS-QTOF is an efficient technique in untargeted metabolomics, avoiding re-injection of the samples for elucidation purposes. In addition, the prediction models for Rt and CCS resulted of help in the elucidation process of potential biomarkers.
    Keywords:  herbal blends smoke; high resolution mass spectrometry; in-silico prediction; ion mobility; omics approaches
  13. Rapid Commun Mass Spectrom. 2021 Apr 20. e9110
      RATIONALE: High mass accuracy is indispensable for reliable identification in MALDI mass spectrometry (imaging). Ubiquitous matrix ions can serve as reference masses for mass calibration, if their sum formula is known. Here we report an overview of ions generated on tissue by eleven common MALDI matrices for use in internal or external mass calibration.METHODS: Matrices covered in this study were applied onto coronal mouse brain sections using a pneumatic sprayer setup. MALDI imaging was performed on a Q Exactive HF orbital trapping mass spectrometer coupled to an AP-SMALDI 10 source. Measurements were conducted with high mass resolution (240k FWHM @ m/z 200) and high mass accuracy with an RMSE of better than 1.5 ppm achieved via internal mass calibration using matrix ions.
    RESULTS: MALDI MS imaging was used to investigate ions generated on tissue by eleven common MALDI matrices. An example of using matrix ions for internal mass calibration in MALDI imaging of drug substances and lipids in murine lung sections is presented. Tables containing the cluster composition, sum formulas as well as the measured and theoretical m/z ratios of the identified ions were compiled for each matrix.
    CONCLUSION: Using matrix ions as reference masses for internal and external mass calibration in MALDI MS imaging is an effective and elegant way to achieve sub ppm mass accuracy as it makes use of ubiquitous signals present in every MALDI MS spectrum without the need for an additional calibration standard.
  14. Food Chem. 2021 Mar 13. pii: S0308-8146(21)00594-X. [Epub ahead of print]357 129588
      Deuterated vitamin D standards are used commonly as internal standards in LC-MS/MS analysis of vitamin D3 and 25-hydroxyvitamin D3 in food. However, the use of various eluent additives, such as methylamine, formic acid and ammonium formate, also contributes to matrix effects and the performance of analysis by affecting accuracy and robustness. For the first time, continuous post-column infusion experiments of isotopically labelled vitamin D3-[d6] were performed to evaluate ion-suppression in a wide variety of food (salmon, cheese, pork fat, pork meat, and egg yolk). Furthermore, results collected using five analytical methods, employing DAD/UV and MS/MS-detectors, were evaluated with in-house and standardised reference materials. The matrix effect was significant when analysing vitamin D3 in most food matrices using the deuterium labelled internal standard. Even though the use of the 13C5-labelled internal standard reduced matrix effects, a standardised method is needed to agree on the true value of vitamin D in food.
    Keywords:  Analogue; Electrospray ionization; Ion suppression; Mass spectrometry; Post-column infusion; Reference material; Vitamin D(3) (PubChem CID: 5280795) 25-hydroxyvitamin D(3) (PubChem CID: 5283731)
  15. J Pharm Biomed Anal. 2021 Apr 06. pii: S0731-7085(21)00177-1. [Epub ahead of print]200 114066
      Short chain fatty acids (SCFAs), generated from microbial fermentation of dietary fibers, can regulate weight, appetite and energy homeostasis. Therefore, measuring SCFAs in fecal samples is important to understand the relationship between dietary patterns, gut microbial metabolism, and their impact on host metabolism homeostasis. However, due to the chemical complexity of fecal samples and the volatility of these SCFAs, the quantitative measurements of SCFAs remain challenging. In this study, we developed an absolute quantitation method for accurate and reliable analysis of SCFAs using an UPLC-Q Exactive HRMS system. Nine C2-C6 SCFAs were first derivatized and then separated on a reversed-phase CSH C18 column, and quantitated by UPLC-HRMS with targeted-selected ion monitoring (t-SIM) mode. Our calibration plots showed high linearity (R2>0.99) with high quantitation accuracy (from 91.24%-118.42%); additional analyses showed excellent precisions ranging from 1.12 % to 6.13 %, and accurate recoveries between 92.38 % and 109.65 % with relative standard deviations of 0.31 %-6.44 %. Meanwhile, the short-term stability, freeze and thaw stability, and 168 h storage stability were tested and reported from 85.07%-106.44% with RSDs 0.44%-20.00%, 98.99%-128.84% with RSDs 0.77%-19.79%, and 77.53%-104.42% with RSDs 0.92%-18.65%, respectively. Lastly, this quantitative method was applied to determine the SCFA concentrations and compositions in forty fecal samples from a group of study subjects participating in an obesity prevention trial, and a broad range of concentrations was noted for the detected SCFAs.
    Keywords:  Dietary fiber; Feces; HRMS; Metabolomics; Short-chain fatty acid
  16. Anal Bioanal Chem. 2021 Apr 05.
      The endocannabinoid system (ECS) is a complex cell-signaling system. To address the growing need of analytics capturing endocannabinoid levels to investigate the ECS, we developed and validated an assay for the quantitative analysis of 14 endocannabinoids and congeners. A simple extraction using protein precipitation with acetonitrile followed by online-trapping high-performance liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) was used to monitor the levels of 14 endocannabinoids in plasma. The assay was validated and intra-run and inter-run accuracies and imprecisions as well as matrix effects, recoveries, and sample stabilities were determined. As a proof of concept, a subset of study samples after naturalistic administration of Cannabis flower and concentrate was analyzed. With the exception of N-oleoyl dopamine and oleamide, all endocannabinoids fulfilled the predefined acceptance criteria. Reproducible recoveries and no significant matrix effects were observed. Sample stability was an issue. Analysis of the proof-of-concept study samples revealed a significantly (p = 0.006) higher concentration of docosatetraenoyl ethanolamide in concentrate users (300 ± 13 pg/mL) compared to flower users (252 ± 11 pg/mL). A robust, sensitive high-throughput assay for the quantitation of 14 endocannabinoids and congeners was successfully validated. Our study showed that it is mandatory to (A) appropriately stabilize samples and (B) separate and separately quantify 1-AG and 2-AG; otherwise, study results are unreliable. The analysis of study samples from Cannabis flower users versus Cannabis concentrate users revealed higher levels of docosatetraenoyl ethanolamide and anandamide (n.s.) in high THC concentrate users in accordance with the existing literature, supporting the validity of the assay measurements. Graphical abstract.
    Keywords:  2-Arachidonoylglycerol; Anandamide; Cannabinoids; Docosatetraenoyl ethanolamide; Endocannabinoid System; Endocannabinoids
  17. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Apr 01. pii: S1570-0232(21)00161-6. [Epub ahead of print]1173 122681
      Okadaic acid (OA) group are diarrheal shellfish poison that accumulates in the midgut glands of shellfish. It is difficult to remove these poisons by normal cooking because they are thermally stable and hydrophobicity. Therefore, in order to prevent foodborne disease due to shellfish poison, analysis by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) before shipment is necessary. Herein the selective analytical method for OA group in shellfish sample using fluorous derivatization coupled with LC-MS/MS was developed. OA group were derivatized with the fluorous alkylamine reagent by condensing agent, and the obtained derivatives were separated with fluorous LC column (Fluofix-II 120E, 250 × 2.0 mm i.d., 5 μm, Fujifilm Wako Pure Chemical). The derivatized OA group were selective retained by fluorous LC column and accurate analysis was enabled. The present method was applied to the analysis of OA and dinophysistoxin-1 (DTX-1) in scallop midgut gland which is the certified reference material provided by national metrology institute of Japan. As a result of analysis using the present method with DTX-2 as the internal standard, the quantitative value were in agreement with the certified value.
    Keywords:  Certified reference material; Dinophysistoxin-1; Fluorous derivatization; LC-MS/MS; Okadaic acid; Scallop midgut gland
  18. Anal Chem. 2021 Apr 19.
      Native mass spectrometry (MS) enables the determination of the molecular mass of protein complexes. Generally, samples for native MS are isolated, purified, and prepared in volatile solutions. However, to understand the function of proteins in living cells, it is essential to characterize the protein complex as is, without isolation/purification of the protein, using the smallest possible amount of the sample. In the present study, we modified the "live single-cell MS" method, which has mainly been used in metabolomics, and applied it to observe hemoglobin directly sampled from human erythrocytes. By optimizing the experimental methods and conditions, we obtained native mass spectra of hemoglobin using only a single erythrocyte, which was directly sampled into a nanoelectrospray ionization emitter using a micromanipulator and microinjector system. That is, our method enables the analysis of ∼0.45 fmol of hemoglobin directly sampled from an erythrocyte. To our knowledge, this is the first report of native MS for endogenous proteins using a single intact human cell.
  19. J Chromatogr Sci. 2021 Apr 19. pii: bmab038. [Epub ahead of print]
      In the present work, tandem dispersive liquid-liquid microextraction (TDLLME) coupled with liquid chromatography was used for the determination of the two antidepressant drugs citalopram (CIT) and sertraline (SER) in complicated matrices. Indeed, the present approach was used to improve the suitability and appropriateness of the dispersive liquid-liquid microextraction (DLLME) method in complicated matrices. Firstly, 10 mL of the sample solution containing the two understudied drugs was extracted into an organic solvent (200 μL of 1,2-dichloromethane) using the DLLME method. Then the extracted analytes were back-extracted into 100 μL of an aqueous acceptor phase. The total extraction time of this method is about 6 min. To achieve the best efficiency for this method, efficient parameters like the kind and volume of the organic solvent and the effect of the ionic strength on the effectiveness of extraction were reviewed and improved. Under the optimized experimental conditions, TDLLME showed good linearity in the range of 100.0-7000.0 ng mL-1. The limits of detection were found to be 10.0 ng mL-1 for CIT and 2.0 ng mL-1 for SER. The relative standard deviation (RSD) is obtained in the range of 0.7-4.1%.
  20. Biomed Chromatogr. 2021 Apr 24. e5146
      Infectious disease such as hepatitis C virus (HCV) is a global clinical issue because of its significant morbidity and mortality. Novel anti-hepatitis C drugs are continuously developed to decrease the pervasiveness of the infection around the world. A synthetic ravidasvir, benzimidazole-naphthylene-imidazole derivatives, has been used as an anti-HCV drug. In this study, the identification of ravidasvir's metabolites and its pharmacokinetics in rats were determined using information-dependent acquisition (IDA) and multiple reaction monitoring (MRM) scanning modes in linear ion trap liquid chromatography tandem-mass (LC-MS/MS) instrument, respectively. Two time-programming linear-gradient chromatographic methods were employed using Kinetex® 2.6 μm C18 (50 × 3 mm) column and Luna® 3 μm HILIC column (100 × 4.6 mm) for the qualitative and quantitative determination of ravidasvir and its metabolites, respectively. In silico prediction where sites in a molecule are susceptible to metabolism by cytochrome P450 was implemented, which helped in proposing the metabolic pathway of ravidasvir. The most dominant metabolite in rat liver microsomal (RLM) samples was oxidative ravidasvir, where one O-demethylated metabolite and eight isomers of the oxidative ravidasvir metabolites were identified. The study provides essential data for proposing the metabolic pathway and successfully applied it to determine the pharmacokinetics of ravidasvir in rat plasma.
    Keywords:  Drug metabolism; Hepatitis C virus (HCV); LC-MS/MS; Linear ion trap; Pharmacokinetics; Ravidasvir
  21. Eur J Mass Spectrom (Chichester). 2021 Apr 04. 14690667211006017
      The approach to quantitative analysis by silicon Surface Assisted Laser Desorption Ionization Mass Spectrometry (Si-SALDI) is proposed. The approach is based on the new method for forming an active surface layer on a silicon substrate by exposing to laser radiation directly in the ion source of a mass spectrometer. The method can be used repeatedly on the same substrate, providing high reproducibility of its surface ionization properties and high ionization efficiency of organic compounds. Within the proposed approach, the methods of improvement of signal reproducibility are also considered, including continuous monitoring of the silicon surface ionization properties using a Knudsen effusion cell; scanning the surface of a silicon substrate with a laser beam; selecting the optimal value of laser fluence and using a reproducible sample introduction technique. It is demonstrated that this approach can be successfully applied to quantify clinically relevant concentrations of pharmaceutical drugs in extracts of blood.
    Keywords:  Surface assisted laser desorption ionization; laser processing; pharmaceutical drugs; quantitative analysis; silicon surface
  22. Anal Chem. 2021 Apr 22.
      Mass spectrometry (MS) can provide high sensitivity and specificity for biochemical assays without the requirement of labels, eliminating the risk of assay interference. However, its use had been limited to lower-throughput assays due to the need for chromatography to overcome ion suppression from the sample matrix. Direct analysis without chromatography has the potential for high throughput if sensitivity is sufficient despite the presence of a matrix. Here, we report and demonstrate a novel direct analysis high-throughput MS system based on infrared matrix-assisted desorption electrospray ionization (IR-MALDESI) that has a potential acquisition rate of 33 spectra/s. We show the development of biochemical assays in standard buffers for wild-type isocitrate dehydrogenase 1 (IDH1), diacylglycerol kinase zeta (DGKζ), and p300 histone acetyltransferase (P300) to demonstrate the suitability of this system for a broad range of high-throughput lead discovery assays. A proof-of-concept pilot screen of ∼3k compounds is also shown for IDH1 and compared to a previously reported fluorescence-based assay. We were able to obtain reliable data at a speed amenable for high-throughput screening of large-scale compound libraries.