bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒02‒16
eight papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Ther Drug Monit. 2020 Feb 12.
      BACKGROUND: Pharmacokinetic studies and therapeutic drug monitoring (TDM) of anticoagulants require a simple, rapid, and reliable analytical method for monitoring plasma concentrations. The aims of the current work were to develop and validate a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of three direct oral anticoagulants (dabigatran, rivaroxaban, and apixaban) in human plasma that is suitable for pharmacokinetic studies and routine TDM in busy hospital laboratories.METHODS: This method included a hydrolysis step to account for the active acylglucuronide metabolites of dabigatran that demonstrate an equivalent anticoagulant effect as dabigatran. After hydrolysis, a simple one-step protein precipitation was used for sample preparation. Total dabigatran (the sum of free dabigatran and the contribution from dabigatran acylglucuronides), rivaroxaban, and apixaban and their corresponding isotopically-labeled internal standards were resolved on a C18(2) column. All compounds were detected using electrospray ionization LC-MS/MS in the positive mode.
    RESULTS: For all three anticoagulants, standard curves were linear over the concentration range of 1.0 to 1000 μg/L (r > 0.99), bias was <±10%, and intra- and inter-day coefficients of variation (imprecision) were <10%. The limit of quantification was 1.0 μg/L. For all three anticoagulants and corresponding isotopically-labeled internal standards, the absolute recoveries were similar and consistent, with mean values of 93%-102%. No significant matrix effects were observed.
    CONCLUSIONS: This method is simple, rapid, robust and reliable, and can be used to analyze the plasma concentrations of the drugs in patients on dabigatran or rivaroxaban therapy.
  2. Mass Spectrom Rev. 2020 Feb 11.
      Mass spectrometry has been the "gold standard" for drugs of abuse (DoA) analysis for many decades because of the selectivity and sensitivity it affords. Recent progress in all aspects of mass spectrometry has seen significant developments in the field of DoA analysis. Mass spectrometry is particularly well suited to address the rapidly proliferating number of very high potency, novel psychoactive substances that are causing an alarming number of fatalities worldwide. This review surveys advancements in the areas of sample preparation, gas and liquid chromatography-mass spectrometry, as well as the rapidly emerging field of ambient ionization mass spectrometry. We have predominantly targeted literature progress over the past ten years and present our outlook for the future. © 2020 Periodicals, Inc. Mass Spec Rev.
    Keywords:  ambient ionization mass spectrometry; drugs of abuse; forensic; gas chromatography-mass spectrometry (GC-MS); liquid chromatography-mass spectrometry (LC-MS); toxicology
  3. J Pharm Biomed Anal. 2020 Jan 09. pii: S0731-7085(19)32361-1. [Epub ahead of print]183 113104
      A novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantify phytosterols (brassicasterol, campesterol, stigmasterol and β-sitosterol) and tocopherols (alpha, beta, gamma and delta) entrapped in the lipid bilayer of a liposomal formulation. Apart from liposomes (a pharmaceutical product), the developed method was able to quantify target analytes in agricultural products, thus showing wide applications. Atmospheric pressure chemical ionization (APCI) was employed due to the enhanced ionization of phytosterols and tocopherols in comparison to electrospray ionization. Unlike published work, the chromatographic conditions were modified to simplify the analytical approach. For the first time, a simple isocratic elution (acetonitrile:methanol 99:1 v/v) was utilized for the separation of four phytosterols and four tocopherols in a single run. A substantially better baseline separation of phytosterols were obtained in comparison to reported methods by using poroshell C18 column. The method has a total run time of 7 min, which is the shortest run time among all reported quantitative methods for the simultaneous determination of four phytosterols and four tocopherols. Calibration curves for all phytosterols were linear in the range of 0.05-10 μg/mL. In the case of tocopherols, alpha tocopherol showed linear response in the range of 0.25-10 μg/mL. However, gamma and delta tocopherols exhibited quadratic relationship in the same concentration range (0.25-10 μg/mL). Validation parameters met the International Conference on Harmonization (ICH) guidelines in terms of selectivity, accuracy, precision, repeatability, sensitivity, matrix effects, dilution integrity and stability. The method was, for the first time, successfully applied for the quantifying phytosterols and tocopherols entrapped inside liposomes. An interesting chromatographic phenomenon was observed during sample analysis. Alpha tocopherol (entrapped in the liposomal lipid bilayer) was found to elute at two retention times, 2.53 min and 3.60 min. Such dual separation was not observed in calibration standards and quality controls. It was concluded that the chiral recognition ability of liposomes made up of phosphatidylcholine separated the enantiomers of alpha tocopherol, giving rise to two peaks at two different retention time. To sum, the reported novel LC-MS/MS method addresses three major analytical shortcomings, namely i)longer run time, ii)complex gradient elution and iii)poor baseline separation of phytosterols and tocopherols.
    Keywords:  APCI; Canola oil deodorizer distillate; LC–MS/MS; Liposomes; Phytosterols; Tocopherols
  4. J Chromatogr Sci. 2020 Feb 11. pii: bmz118. [Epub ahead of print]
      Two high-performance liquid chromatography-diode array detection methods have been developed and validated for the simultaneous quantification of genistein (GNS) and all trans retinoic acid (ATRA) as a novel anticancer combination therapy in their co-formulated nanoparticles and in rat plasma. Separation was performed on C18 column (250 × 4.6 mm, 5 μm) using celecoxib as internal standard. A mobile phase containing acetonitrile and water adjusted to pH 3 using 1% trifluoroacetic acid was delivered in gradient elution modes with time programmed UV detection. For extraction of the drugs and the internal standard from rat plasma, liquid- liquid extraction was applied. The proposed methods were validated as per International Conference on Harmonisation (ICH) guidelines (in the range 0.1-10 μg/mL for analysis of GNS and ATRA in nanoparticles) or according to Food and Drug Administration (FDA) guidance on bioanalytical method validation (in the range 0.025-20 μg/mL for analysis of GNS and ATRA in rat plasma). Pharmacokinetic study in six rats was performed following intravenous (IV) administration of a single dose of 0.5 mg/Kg of GNS and ATRA. The drugs' concentrations were measured up to 24 hours, and different pharmacokinetic parameters were calculated. The obtained parameters were comparable with the reported values for IV administration of each drug alone in rats. This confirms the applicability of the proposed method in monitoring the levels of the two drugs in vivo following their coadministration and indicating that the two drugs could be coadministered as a promising novel combination therapy for the treatment of lung cancer without great alteration in their pharmacokinetic parameters compared with their individual IV administration.
  5. Metabolites. 2020 Feb 12. pii: E66. [Epub ahead of print]10(2):
      Elementary Flux Modes (EFMs) are a tool for constraint-based modeling and metabolic network analysis. However, systematic and automated visualization of EFMs, capable of integrating various data types is still a challenge. In this study, we developed an extension for the widely adopted COBRA Toolbox, EFMviz, for analysis and graphical visualization of EFMs as networks of reactions, metabolites and genes. The analysis workflow offers a platform for EFM visualization to improve EFM interpretability by connecting COBRA toolbox with the network analysis and visualization software Cytoscape. The biological applicability of EFMviz is demonstrated in two use cases on medium (Escherichia coli, iAF1260) and large (human, Recon 2.2) genome-scale metabolic models. EFMviz is open-source and integrated into COBRA Toolbox. The analysis workflows used for the two use cases are detailed in the two tutorials provided with EFMviz along with the data used in this study.
    Keywords:  Elementary Flux Modes; Genome-Scale Metabolic Models; SBML; data visualization; network visualization
  6. Phytochem Anal. 2020 Feb 11.
      INTRODUCTION: An efficient characterisation of metabolites is a crucial task in many aspects of basic research, such as the de-replication of crude extracts in natural products chemistry or the tentative identification of compounds in untargeted metabolomics.OBJECTIVE: The goal of this work is the evaluation of the reaction with phenylhydrazine for the derivatisation post-column in situ of carbonyl-containing compounds in liquid chromatography-mass spectrometry (LC-MS).
    MATERIALS AND METHODS: LC-MS was performed using electrospray, Atmospheric Pressure Chemical Ionisation (APCI) or Atmospheric Pressure Photoionization (APPI) as ionisation techniques. The post-column addition of phenylhydrazine was done through a syringe pump via a T-junction before entrance to the ion source.
    RESULTS: A variety of natural products having carbonyl groups, such as cycloartanes, steroids, cardenolides and other terpenoids, were analysed by this method. In the case of compounds with non-hindered aldehyde or keto groups, the main signals of the mass spectra were those corresponding to the phenylhydrazones. However, the spectra of compounds with hindered carbonyl groups displayed mainly those signals corresponding to the product of the nucleophilic addition adduct of phenylhydrazine to the carbonyl, which is the first step of the derivatisation process. Finally, those compounds with conjugated ketones did not react with phenylhydrazine. This methodology was applied in the analysis of crude natural extracts.
    CONCLUSION: The results show that in situ derivatisation of carbonyl compounds in the ionisation source was achieved, yielding the typical derivatives of carbonyl compounds with phenylhydrazine.
    Keywords:  LC-MS; carbonyl compounds; derivatisation procedure; phenylhydrazones
  7. Sci Adv. 2020 01;6(4): eaay5206
      Noninvasive blood glucose monitoring has been a long-standing dream in diabetes management. The use of Raman spectroscopy, with its molecular specificity, has been investigated in this regard over the past decade. Previous studies reported on glucose sensing based on indirect evidence such as statistical correlation to the reference glucose concentration. However, these claims fail to demonstrate glucose Raman peaks, which has raised questions regarding the effectiveness of Raman spectroscopy for glucose sensing. Here, we demonstrate the first direct observation of glucose Raman peaks from in vivo skin. The signal intensities varied proportional to the reference glucose concentrations in three live swine glucose clamping experiments. Tracking spectral intensity based on linearity enabled accurate prospective prediction in within-subject and intersubject models. Our direct demonstration of glucose signal may quiet the long debate about whether glucose Raman spectra can be measured in vivo in transcutaneous glucose sensing.
  8. Transl Clin Pharmacol. 2018 Sep;26(3): 134-140
      This study aimed to develop a UPLC-MS/MS method for determining plasma levels of L-aspartic acid and L-asparagine and the activity of L-asparaginase. L-aspartic acid, L-asparagine, and L-aspartic acid-2,3,3-d3 were extracted from human plasma by protein precipitation with sulfosalicylic acid (30%, v/v). The plasma samples were analyzed using an Imtakt Intrada amino acid analysis column with 25 mM ammonium formate and 0.5% formic acid in acetonitrile as the mobile phase with step gradient method at a flow rate of 0.5 mL/min. The injection volume was 5 µL, and the total run time was 15 min. Inter- and intra-batch accuracies (%) ranged from 96.62-106.0% for L-aspartic acid and 89.85-104.8%, for L-asparagine, and the coefficient of variation (CV%) did not exceed 7%. The validation results for L-aspartic acid and L-asparagine satisfied the specified criterion, however, the results for L-asparaginase activity assay showed a borderline validity. This study could be a foundation for further development of therapeutic drug monitoring systems using UPLC-MS/MS.
    Keywords:  L-asparaginase; L-asparagine; L-aspartic acid; UPLC-MS/MS