bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒12‒20
sixteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Metabolomics. 2020 Dec 15. 16(12): 128
      INTRODUCTION: Metabolomics studies are not routine when quantifying amino acids (AA) in congenital heart disease (CHD).OBJECTIVES: Comparative analysis of 24 AA in serum by traditional high-performance liquid chromatography (HPLC) based on ion exchange and ninhydrin derivatisation followed by photometry (PM) with ultra-high-performance liquid chromatography and phenylisothiocyanate derivatisation followed by tandem mass spectrometry (TMS); interpretation of findings in CHD patients and controls.
    METHODS: PM: Sample analysis as above (total run time, ~ 119 min). TMS: Sample analysis by AbsoluteIDQ® p180 kit assay (BIOCRATES Life Sciences AG, Innsbruck, Austria), which employs PITC derivatisation; separation of analytes on a Waters Acquity UHPLC BEH18 C18 reversed-phase column, using water and acetonitrile with 0.1% formic acid as the mobile phases; and quantification on a Triple-Stage Quadrupole tandem mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with electrospray ionisation in the presence of internal standards (total run time, ~ 8 min). Calculation of coefficients of variation (CV) (for precision), intra- and interday accuracies, limits of detection (LOD), limits of quantification (LOQ), and mean concentrations.
    RESULTS: Both methods yielded acceptable results with regard to precision (CV < 10% PM, < 20% TMS), accuracies (< 10% PM, < 34% TMS), LOD, and LOQ. For both Fontan patients and controls AA concentrations differed significantly between methods, but patterns yielded overall were parallel.
    CONCLUSION: Serum AA concentrations differ with analytical methods but both methods are suitable for AA pattern recognition. TMS is a time-saving alternative to traditional PM under physiological conditions as well as in patients with CHD.
    TRIAL REGISTRATION NUMBER: Identifier NCT03886935, date of registration March 27th, 2019 (retrospectively registered).
    Keywords:  Amino acid metabolism; Congenital heart disease; Fontan; Metabolomics; Pattern recognition; Tandem mass spectrometry
  2. J Pharm Biomed Anal. 2020 Dec 02. pii: S0731-7085(20)31701-5. [Epub ahead of print] 113815
      (S)-1-(5-(4-Methylpiperazin-1-yl)-2,4-dinitrophenyl)pyrrolidine-2-carboxylic acid (Pro-PPZ) was employed as a chiral derivatization reagent (CDR) for the efficient enantioseparation and ultrasensitive mass spectrometric detection of chiral amines. Pro-PPZ was prepared from the one-step reaction of 1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ) and l-proline. Two amines and two amino acid methyl esters were selected as model chiral amines, which were easily labeled with Pro-PPZ under mild reaction conditions (35 °C for 10 min) generating Pro-PPZ-amine derivatives. The resulting diastereomers were completely separated by reversed-phase liquid chromatography (RP-LC) using an ODS column (Rs = 3.4-17.0 for amines). Ultrasensitive detection limits on femtomolar level were obtained for the tested amines using multiple reaction monitoring (MRM) chromatograms at a single monitoring ion, m/z 289 (0.1-5.0 fmol for amines). The practical metabolite analysis of (R)-1-aminoindan (R-AI) in saliva samples was performed by LC-MS/MS using the Pro-PPZ derivatization method. The method was validated in terms of precision, accuracy, and linearity. Using this method, R-AI concentrations in saliva were determined after a single oral administration of the drug rasagiline to healthy male and female subjects, but no (S)-1-aminoindan (S-AI) was detected, which suggesting that R-AI was not converted into S-enantiomer in the metabolic process. R-AI concentrations in four healthy volunteers ranged from 32.85 nM to 49.45 nM, with an average value of 43.76 nM. To date, there is no LC-MS (or MS/MS) method reported for the enantioselective determination of R-AI in human saliva samples.
    Keywords:  Chiral amines; Chiral derivatization reagent; Mass spectrometry; Saliva analysis
  3. Electrophoresis. 2020 Dec 17.
      Non-targeted analysis of water samples using liquid chromatography combined with high-resolution mass spectrometers is an emerging approach for surface water monitoring and evaluation of water treatment processes. In this study, sample pre-concentration via direct, large volume injection with 500 μL and 1000 μL injection volumes was compared to SPE regarding analytical performance parameters in targeted and non-targeted workflows. In targeted analysis, the methods were evaluated in terms of LOD and intra-batch precision of the selected compounds, whereas in non-targeted analysis, the number of detected unknown compounds, the method´s intra-batch precision and the retention time vs. molecular mass pattern of the detected unknowns were evaluated. In addition, a novel intensity drift correction method was developed that is not based on QC samples and makes use of the signals obtained for continuously infused reference compounds, which are conventionally utilized for on-line mass drift correction. It could be demonstrated that the new correction method significantly reduced the bias introduced by instrumental drift and is important for the reliable inter-comparison of different non-targeted methods. Inter-comparison of results showed that the 1000 μL large volume injection method revealed the best performance in terms of precision under repeatability conditions of measurement as well as lower LODs for targeted compound analysis. In non-targeted analysis, the SPE method detected a higher number of unknown compounds but exhibited also a higher uncertainty of measurement caused by matrix effects. This article is protected by copyright. All rights reserved.
    Keywords:  High resolution mass spectrometry; Large volume injection; Natural waters; Non-targeted analysis; Signal drift correction
  4. J Chromatogr A. 2020 Dec 03. pii: S0021-9673(20)31056-6. [Epub ahead of print]1636 461782
      Cytokinins (CKs) are one class of important phytohormones widely investigated in most aspects of plant life. Similar to other phytohormones, CKs and their glycoconjugates are hydrophilic. Their ionization efficiencies for mass spectrometry (MS) detection are rather poor, whereas their retention and separation on reverse phase liquid chromatography (RPLC) are often unsatisfying. Chemical isotope labelling LC-MS analysis methods have been developed for most other phytohormones, enhancing their LC separations and quantitative sensitivity. However, there are currently no reports for chemical-labelled CKs. Here, we report a new chemical isotope labelling LC-MS analytical method for one-pot derivatization of CK bases and their glycoconjugates, based on differential benzylation labelling of the adenine skeleton of CKs with benzyl bromide and its deuterium isotope-labelled reagent. Benzylation alters the hydrophilicity of CKs and their glycoconjugates, improving their retention and separation on RPLC. The developed method demonstrated enhanced sensitivity, as the CKs and their glycoconjugates could be analysed with LODs within the range of 0.62-25.9 pg/mL. The method also demonstrated good intra- and inter-day precisions with standard deviations in the range of 1.9%-13.0%, and acceptable accuracy with recoveries in the range of 84.0%-119.9%. The developed method was employed on the quantitation of CKs in the fresh roots of Astragalus membranaceus collected from both fertilized and unfertilized fields. The significant impact that fertilizers had on endogenous CKs metabolism was observed. As such, monitoring endogenous CKs and their metabolites might be promising to control fertilizer abuse.
    Keywords:  Adenine; Benzyl bromide; Chemical isotope labelling; Cytokinins; LC-MS; Phytohormones
  5. Electrophoresis. 2020 Dec 12.
      Gangliosides are particularly abundant in the nervous system (NS) where their pattern and structure in a certain milieu or a defined region exhibit a pronounced specificity. Since gangliosides are useful biomarkers for diagnosis of NS ailments, a clear-cut mapping of individual components represents a prerequisite for designing ganglioside-based diagnostic procedures, treatments or vaccines. These bioclinical aspects and the high diversity of ganglioside species claim for development of specific analytical strategies. This review summarizes the state-of-the-art in the implementation of separation techniques and microfluidics coupled to mass spectrometry (MS), which have contributed significantly to the advancement of the field. In the first part, the review discusses relevant approaches based on high performance liquid chromatography (HPLC) MS and capillary electrophoresis (CE) coupled to electrospray (ESI) MS and their applications in the characterization of gangliosides expressed in healthy and diseased NS. A considerable section is dedicated to microfluidics MS and ion mobility separation (IMS) MS, developed for the study of brain gangliosidome and its changes triggered by various factors, as well as for ganglioside biomarker discovery in neurodegenerative diseases and brain cancer. In the last part of the review, the benefits and perspectives in ganglioside research of these high performance techniques are presented. This article is protected by copyright. All rights reserved.
    Keywords:  capillary electrophoresis; electrospray ionization mass spectrometry; gangliosides; high performance liquid chromatography; ion mobility mass spectrometry; microfluidics
  6. J Chromatogr Sci. 2020 Dec 19. pii: bmaa105. [Epub ahead of print]
      An high performance liquid chromatography (HPLC) method, applying diode array (DAD) and coulometric array detection simultaneously, was developed to determine monomeric phenolic compounds in lignin hydrolysates. To allow for additional mass spectrometric detection, a formic acid gradient was designed as an alternative to the non-volatile phosphate buffer gradient. Within a total run time of 28 min, 10 out of 14 analytes were baseline separated by the formic acid gradient, and 8 compounds were completely resolved by the phosphate gradient. The dependence of the coulometric detection on cell potential, potential step width, eluent pH and eluent composition was comprehensively tested. Detection limits ≤20 μg/L were achieved by DAD detection for nine analytes and by coulometric array detection for all analytes, demonstrating the superior sensitivity of electrochemical detection. DAD offered better linearity and reproducibility. Inter-day precision of peak heights spanned from 0.26 to 2.00% (formic acid gradient) and from 0.75 to 2.85% (phosphate buffer gradient) operating the DAD detector, but it exceeded 10% for several compounds applying the coulometric array detector. The simultaneous operation of both detectors offered an enhanced certainty of substance identification. The appropriateness of the method was confirmed by the analysis of various hydrolysates of lignin-containing materials.
  7. Res Pharm Sci. 2020 Aug;15(4): 312-322
      Background and purpose: Lonidamine is a hexokinase II inhibitor, works as an anticancer molecule, and is extensively explored in clinical trials. Limited information prevails about the stability-indicating methods which could determine the forced degradation of lonidamine under stressed conditions. Hence, we report the use of a rapid, sensitive, reproducible, and highly accurate liquid chromatography and mass spectrometry method to analyze lonidamine degradation.Experimental approach: The Xbridge BEH shield reverse phase C18 column (2.5 μm, 4.6 × 75 mm) using isocratic 50:50 water: acetonitrile with 0.1% formic acid can detect lonidamine with help of mass spectrometer in tandem with an ultraviolet (UV) detector at 260 nm wavelength.
    Findings/ Results: A linear curve with r2 > 0.99 was obtained for tandem liquid chromatography-mass spectrometry (LC-MS)-UV based detections. This study demonstrated (in the present set up of isocratic elution) that LC-MS based detection has a relatively high sensitivity (S/N (10 ng/mL): 220 and S/N (20 ng/mL): 945) and accuracy at lower detection and quantitation levels, respectively. In addition to developing the LC-MS method, we also report that the current method is stability-indicating and shows that lonidamine gets degraded over time under all three stress conditions; acidic, basic, and oxidative.
    Conclusion and implications: LC-MS based quantitation of lonidamine proved to be a better method compared to high-performance liquid chromatography (HPLC)-UV detections for mapping lonidamine degradation. This is the first report on the stability-indicating method for studying the forced degradation of lonidamine using LC-MS method.
    Keywords:  Forced degradation; LC-MS; Lonidamine; Stability indicating
  8. J Sep Sci. 2020 Dec 19.
      Solid-substrate electrospray ionization MS is an important ambient ionization technology to simplify MS analysis. Nowadays, its separation application has been reported increasingly, however, the detailed separation mechanism is still indistinct although the chromatographic effect was reported as a possible factor. In this study, substrate-filled capillary electrospray ionization MS was developed as an ideal model to investigate the separation mechanism using over thirty small molecules (neutral, basic, and weakly acidic) as model compounds with C18-bonded silica gel and silica gel as the substrates. The chromatographic effect was validated by the negative t-value of oil-water distribution coefficient, and the electric field effect was verified by the paired t-test (p < 0.01) between the retention times at 5.5 kV and 4.0 kV. A differential equation was proposed to quantify the compound retention under electric field. The quantitative method was validated to rapidly quantify proline (31.88 μg/mL) and hydroxyproline (20.71 μg/mL) in plasma with acceptable selectivity and accuracy. In conclusion, the separation mechanism of solid-substrate electrospray ionization MS was the combination of the chromatographic and electric field effects, which could provide theoretical guidance for the separation optimization, and also promote its applications in biological, pharmaceutical, forensic, food and environmental analyses, etc. This article is protected by copyright. All rights reserved.
    Keywords:  chromatographic effect; electric field effect; electroosmosis control; separation mechanism; solid-substrate electrospray ionization
  9. Sci Rep. 2020 Dec 15. 10(1): 22008
      The noninvasive diagnosis and monitoring of high prevalence diseases such as cardiovascular diseases, cancers and chronic respiratory diseases are currently priority objectives in the area of health. In this regard, the analysis of volatile organic compounds (VOCs) has been identified as a potential noninvasive tool for the diagnosis and surveillance of several diseases. Despite the advantages of this strategy, it is not yet a routine clinical tool. The lack of reproducible protocols for each step of the biomarker discovery phase is an obstacle of the current state. Specifically, this issue is present at the data preprocessing step. Thus, an open source workflow for preprocessing the data obtained by the analysis of exhaled breath samples using gas chromatography coupled with single quadrupole mass spectrometry (GC/MS) is presented in this paper. This workflow is based on the connection of two approaches to transform raw data into a useful matrix for statistical analysis. Moreover, this workflow includes matching compounds from breath samples with a spectral library. Three free packages (xcms, cliqueMS and eRah) written in the language R are used for this purpose. Furthermore, this paper presents a suitable protocol for exhaled breath sample collection from infants under 2 years of age for GC/MS.
  10. Food Chem. 2020 Dec 03. pii: S0308-8146(20)32636-4. [Epub ahead of print]345 128774
      A rapid, low-cost, and selective method for simultaneous and direct determination of maneb group residues (containing ethylenebis and propylenebis dithiocarbamates) in fruit by liquid chromatography coupled to tandem mass spectrometry was developed and validated in the current study. The results showed the maneb group could be melt and stabilized by 5 v% ethylenediamine for 60 days keeping in conventional refrigerators, in which a stable and ionizable pentadentate ligand complex was considered to be formed by the bidentate diamine and sulfhydryl followed by Density Functional Theory calculation. The validated method showed a sensitive quantification limits (0.03 mg/kg), a steady recovery (82.1%-91.0%) and an excellent precision (2.7%-4.3% RSD). This method is applied to analyze fruit samples and achieved satisfactory results. Therefore, this method can be proposed as a robust analytical method of maneb group in fruit, and can be adapted to detect other compounds with sulfhydryl group.
    Keywords:  Direct-determination; High performance liquid chromatography-tandem mass spectrometry; Maneb group; Stabilization
  11. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 30. pii: S1570-0232(20)31342-8. [Epub ahead of print]1162 122466
      Deriving from targeted kinase inhibitors (TKIs), targeted covalent kinase inhibitors (TCKIs) are a new class of TKIs that are covalently bound to their target residue of kinase receptors. Currently, there are many new TCKIs under clinical development besides afatinib, ibrutinib, osimertinib, neratinib, acalabrutinib, dacomitinib, and zanubrutinib that are already approved by the FDA. Subsequently, there is an increasing demand for bioanalytical methods to qualitatively and quantitively investigate those compounds, leading to a number of papers reporting the development, validation, and use of bioanalytical methods for TCKIs. Most publications describe the technological set up of analytical methods that allow quantification of TCKIs in various biomatrices such as plasma, cerebrospinal fluid, urine, tissue, and liver microsomes. In addition, the identification of metabolites and biotransformation pathways of new TCKIs has gained more interest in recent years. We provide an overview of bioanalytical methods of this new class of TCKIs. The included issues are sample pretreatment, chromatographic separation, detection, and method validation. In the scope of bioanalysis of TCKIs, protein precipitation is mostly applied to treat the biological matrices sample. Liquid chromatographic in reversed-phase mode (RPLC) and mass detection with triple quadrupole (QqQ) are the most often utilized separation and quantitative detection modes, respectively. There may be a possibility of increased use of the high-resolution mass spectrometry (HRMS) for qualitative investigation purposes in the future. We also found that US FDA and EMA guidelines are the most common guidelines employed as validation framework for the bioanalytical methods of TCKIs.
    Keywords:  Biomatrices; Chromatography; Covalent targeted kinase inhibitors; Mass spectrometry; Metabolites
  12. Molecules. 2020 Dec 09. pii: E5799. [Epub ahead of print]25(24):
      A new approach for the sensitive, robust and rapid determination of idarubicin (IDA) in human plasma and urine samples based on liquid chromatography with fluorescence detection (LC-FL) was developed. Satisfactory chromatographic separation of the analyte after solid-phase extraction (SPE) was performed on a Discovery HS C18 analytical column using a mixture of acetonitrile and 0.1% formic acid in water as the mobile phase in isocratic mode. IDA and daunorubicin hydrochloride used as an internal standard (I.S.) were monitored at the excitation and emission wavelengths of 487 and 547 nm, respectively. The method was validated according to the FDA and ICH guidelines. The linearity was confirmed in the range of 0.1-50 ng/mL and 0.25-200 ng/mL, while the limit of detection (LOD) was 0.05 and 0.125 ng/mL in plasma and urine samples, respectively. The developed LC-FL method was successfully applied for drug determinations in human plasma and urine after oral administration of IDA at a dose of 10 mg to a patient with highly advanced alveolar rhabdomyosarcoma (RMA). Moreover, the potential exposure to IDA present in both fluids for healthcare workers and the caregivers of patients has been evaluated. The present LC-FL method can be a useful tool in pharmacokinetic and clinical investigations, in the monitoring of chemotherapy containing IDA, as well as for sensitive and reliable IDA quantitation in biological fluids.
    Keywords:  anthracyclines; drug monitoring; human and urine samples; idarubicin; liquid chromatography with fluorescence detection (LC-FL); liquid-liquid extraction (LLE); solid-phase extraction (SPE); solid-phase microextraction (SPME)
  13. Can Commun Dis Rep. 2020 Oct 01. 46(10): 333-338
      Background: The use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) systems for bacterial identification has rapidly become a front line tool for diagnostic laboratories, superseding classical microbiological methods that previously triggered the identification of higher risk pathogens. Unknown Risk Group 3 isolates have been misidentified as less pathogenic species due to spectral library availability, content and quality. Consequently, exposure to higher risk pathogens has been reported within Canadian laboratory staff following the implementation of MALDI-TOF MS. This overview aims to communicate the potential risk to laboratory staff of inaccurate identification of security-sensitive biological agents (SSBA) bacteria and to provide suggestions to mitigate.Methods: Cultures were manipulated in a Biosafety Level 3 laboratory, prepared for MALDI-TOF MS analysis via full chemical extraction and analysed on a Bruker Microflex LT instrument. Data were analyzed with Biotyper software; comparing raw spectra against MS profiles in three libraries: Bruker Taxonomy; Bruker Security-Restricted; and National Microbiology Laboratory (NML) SSBA libraries. Four years of Bruker MALDI-TOF MS data acquired in-house were reviewed.
    Results: In general, the Bruker MS spectral libraries were less successful in identifying the SSBA bacteria. More successful was the NML library. For example, using a high score cut-off (greater than 2.0), the Bruker SR library was unable to identify 52.8% of our Risk Group 3 agents and near neighbours to the species-level with confidence, whereas the custom NML library was unable to identify only 20.3% of the samples.
    Conclusion: The last four years of data demonstrated both the importance of library selection and the limitations of the various spectral libraries. Enhanced standard operating procedures are advised to reduce laboratory exposure to SSBAs when using MALDI-TOF MS as a front line identification tool.
    Keywords:  MALDI-TOF mass spectrometry; bacterial pathogens; clinical microbiology laboratory diagnostics; security-sensitive
  14. Food Chem. 2020 Dec 08. pii: S0308-8146(20)32697-2. [Epub ahead of print]345 128835
      Growing evidence of vitamin K's importance in human health beyond blood coagulation and bone health necessitates its further research. A method involving extraction, lipase treatment, clean-up, and detection and quantification by LC-ESI-MS/MS of phylloquinone (PK), menaquinone-4 (MK-4), menaquinone-7 (MK-7) and menaquinone-9 (MK-9) was developed, and single-laboratory validated. The matrices included in the validation were hazelnut, cheese, broccoli, and pork. The LC-method runtime was 9 min. The LOQ for PK, MK-4 and MK-7 was 0.5 µg/100 g food, while for MK-9 it was 2.5 µg/100 g food. The intra- and inter-day precision was <15% for endogenous and spiked levels, except for low content at 4 times the LOQ. Trueness was assessed to be in the range 94-125% for spiking at levels approximately 4 and 10 times LOQ. It is further shown that deuterium labelled MK-7 can be used as an internal standard for MK-9.
    Keywords:  LC-ESI-MS/MS; Matrix effect; Menaquinones; Method validation; Phylloquinone
  15. Anal Chem. 2020 Dec 18.
      A fast and fully automated method for chiral analysis has been developed by combining a chiral derivatization approach with high-resolution trapped ion mobility separation. Although the presented approach can be potentially applied to diverse types of chiral compounds, several benchmark amino acids were used as model compounds, focusing on the smallest amino acid alanine. An autosampler with an integrated chromatography system was used for inline chiral derivatization with (S)-naproxen chloride and subsequent preseparation. Afterwards, derivatized amino acids were directly introduced into the electrospray interface of a trapped ion mobility-mass spectrometer for rapid diastereomer separation in the gas phase. This unique combination of preseparation and trapped ion mobility spectrometry separation in the negative ion mode enabled rapid chiral analysis within 3 min per sample. Furthermore, the diastereomer separation proved to be independent of alkali salts or other metal ions, offering robustness with regard to samples containing high amounts of salts. Highly sensitive detection of amino acid diastereomers was possible down to the lower nanomolar concentration range, and enantiomeric ratios could be readily determined from the recorded mobilograms with excellent reproducibility and precision. To demonstrate the general applicability of our method, alanine and other amino acids were analyzed from soy sauces and seasonings, which revealed extraordinarily high d-Ala contents of up to 99% in all samples.
  16. Front Pharmacol. 2020 ;11 574638
      Pinostrobin is a natural flavonoid found in various plants, well known for its wide range of pharmacological activities. However, there are few reports regarding the pharmacokinetics, tissue distribution, metabolism, and excretion of pinostrobin in rats after oral administration as a single compound. Therefore, we established a method using ultra-high-performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry (UPLC-LTQ orbitrap-MS/MS) to determine pinostrobin and its metabolites in rat plasma, urine, feces, bile, and tissue homogenates. Pharmacokinetic parameters were measured. The large apparent volume of distribution implied that pinostrobin preferentially bound to tissues and preferably remained within the body. Based on previous pharmacological studies of its antiulcer, anti-HP, anti-inflammatory, and antioxidant activities, pinostrobin is mostly distributed in the gastrointestinal tract, indicating its potential as an effective component of traditional Chinese medicines for the treatment of peptic ulcers. Furthermore, 30 flavonoid metabolites were screened using UPLC-LTQ orbitrap-MS/MS. The metabolism pathways (mainly hydroxylation, demethylation, glucuronidation, and sulfation) of pinostrobin in rats have also been proposed. A small amount of pinostrobin in its parent form is excreted through the urine, feces, and bile, indicating that it is mainly metabolized in vivo. In this study, we systemically investigated the pharmacokinetics, tissue distribution, metabolism, and excretion of pinostrobin in rats. Our results provide a significant basis for the clinical development and application of pinostrobin as well as traditional Chinese medicines containing pinostrobin.
    Keywords:  UPLC-LTQ orbitrap-MS/MS; excretion; metabolites; pharmacokinetics; pinostrobin; tissue distribution