bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022–02–13
nineteen papers selected by
Sofia Costa, Icahn School of Medicine at Mount Sinai
  1. A simple and reliable bile acid assay in human serum by LC-MS/MS.
  2. Maximizing MS/MS Acquisition for Lipidomics Using Capillary Separation and Orbitrap Tribrid Mass Spectrometer.
  3. Clinical application of liver diseases diagnosis using ultrahigh-sensitive liquid chromatography-mass spectrometry for sialic acids detection.
  4. A protocol for investigating lipidomic dysregulation and discovering lipid biomarkers from human serums.
  5. Comparative metabolomics with Metaboseek reveals functions of a conserved fat metabolism pathway in C. elegans.
  6. MultiConditionRT: Predicting liquid chromatography retention time for emerging contaminants for a wide range of eluent compositions and stationary phases.
  7. A Practitioner's Dilemma Mass Spectrometry-Based Annotation and Identification of Human Plasma and Urinary Polyphenol Metabolites.
  8. Biological matrix compatible porous thin film for quick extraction of drugs of abuse from urine prior to liquid chromatography-mass spectrometry analysis.
  9. Simultaneous Determination of Vitamin D and Its Hydroxylated and Esterified Metabolites by Ultrahigh-Performance Supercritical Fluid Chromatography-Tandem Mass Spectrometry.
  10. A selective and accurate liquid chromatography-tandem mass spectrometry method for the quantitation of the novel 5-HT4 receptor partial agonist SUVN-D4010 (Usmarapride) in human plasma and urine.
  11. Development and application of a highly sensitive LC-MS/MS method for simultaneous quantification of N,N-dimethyltryptamine and two of its metabolites in human plasma.
  12. Method development and validation for simultaneous determination of six tyrosine kinase inhibitors and two active metabolites in human plasma/serum using UPLC-MS/MS for therapeutic drug monitoring.
  13. Noise-Reduced Quantitative Fluorine NMR Spectroscopy Reveals the Presence of Additional Per- and Polyfluorinated Alkyl Substances in Environmental and Biological Samples When Compared with Routine Mass Spectrometry Methods.
  14. A method for a comprehensive lipidomic analysis of flaxseed (Linum usitatissimum) with the use of LC-Q-TOF-MS and dispersive micro-solid-phase (μDSPE) extraction.
  15. Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for erythrocyte phosphatidylethanol revealing critical considerations for its use as a clinical biomarker.
  16. Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments.
  17. Development of a new Ultra-High-Performance liquid Chromatography-Tandem mass spectrometry (UHPLC-MS/MS) method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay.
  18. Advancement in Analytical Strategies for Quantification of Biomarkers with a Special Emphasis on Surrogate Approaches.
  19. Metaboprep: an R package for pre-analysis data description and processing.
  1. J Clin Lab Anal. 2022 Feb 09. e24279
    A simple and reliable bile acid assay in human serum by LC-MS/MS.
    Ying Shen, Ke Liu, Xia Luo, Qing Guan, Liming Cheng.
       BACKGROUND: Bile acids, as important signaling molecules and regulatory factors acting on glucose, lipid, and energy metabolism, are always involved in liver, biliary, and intestinal diseases. Development and validation of a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of bile acids is significant for the routine clinical testing.
    METHODS: Fifty microlitre of serum was mixed with 10 μl of the internal standard working solution and then 140 μl of methanol for protein precipitation. After centrifuged, the supernatant was directly used for LC-MS/MS analysis.
    RESULTS: Good separation of all bile acid species was achieved. The method was validated with consistent linearity for individual bile acids, good recovery, low carryover, satisfactory sample stability, and analytical specificity against hemolysis, lipemia, and bilirubinemia. The intra-day and the inter-day imprecision values were in the range of 1.53%-10.63% and 3.01%-13.98%, respectively. No obvious matrix effect was observed. The reference intervals of bile acids in adults have been established for the clinical testing.
    CONCLUSIONS: The low sample volume, simple sample preparation, good separation of all species, and satisfying validation results make this LC-MS/MS approach suitable for usage as a high-throughput assay in routine clinical laboratories.
    Keywords:  LC-MS/MS; bile acids; clinical testing; serum
    DOI:  https://doi.org/10.1002/jcla.24279
  2. Anal Chem. 2022 Feb 09.
    Maximizing MS/MS Acquisition for Lipidomics Using Capillary Separation and Orbitrap Tribrid Mass Spectrometer.
    Yuchen He, Dain R Brademan, Paul D Hutchins, Katherine A Overmyer, Joshua J Coon.
      Liquid chromatography-mass spectrometry (LC-MS) is a typical strategy for lipidomics analysis. Although capillary LC-MS is a common analytical technique for proteomics analysis, its application to lipidomics has been limited. In this study, we aim at improving lipid identifications achieved in a single LC-MS analysis by a 3-fold approach: capillary LC and nanoelectrospray for enhanced ionization, ion trap for higher sensitivity tandem MS, and parallelization of mass analyzers for increased speed of acquisition on an Orbitrap hybrid system. By applying the methods to a complex lipid mixture of human plasma, we identified and performed relative quantification on over 1500 lipids within a 60 min capillary LC-MS analysis.
    DOI:  https://doi.org/10.1021/acs.analchem.1c05552
  3. J Chromatogr A. 2022 Jan 20. pii: S0021-9673(22)00035-8. [Epub ahead of print]1666 462837
    Clinical application of liver diseases diagnosis using ultrahigh-sensitive liquid chromatography-mass spectrometry for sialic acids detection.
    Juan Li, Ting Wu, Xiucui Zhang, Yiping Du, Bo Wei, Junxue Wang.
      Sialic acids are a group of nine-carbon N-acetylated derivatives of neuraminic acid containing a keto group at position C2 (ketononose), which play important roles in many biological processes. The simultaneous detection of free sialic acid (FSA) and total sialic acid (TSA) is always challenging due to three orders of magnitude difference. An accurate and robust chemical derivatization-based liquid chromatography-mass spectrometry (LC-MS) method was proposed here to quantify both TSA and FSA in human serum with only 1 μL human serum consumption. The derivatization method with Girard's P (GP) reagent provided an ultrasensitive analysis of sialic acids as only [GP+SA-H2O]+ ions derivatized from SA could be detected by LC-MS. The limit of quantification (LOQ) of SA was less than 5 fg (S/N = 47), which was the most sensitive measurement published for SAs in biomatrices. In addition, no matrix effect existed after 10000-fold dilution of serum extracts. The recovery rates were in the range of 98.1-114.0% and the coefficient of variations (CV) was within 5%. The method has been successfully applied for the quantification of TSA and FSA in serums of patients with different liver diseases. The specificities of TSA and FSA for the early diagnosis of severe hepatopathies were higher than most of the lab blood test indicators with area under the curve (AUC) of 0.900 and 0.882.
    Keywords:  Girard's P reagent derivatization; Human serum; LC-MS; Liver diseases; Sialic acids
    DOI:  https://doi.org/10.1016/j.chroma.2022.462837
  4. STAR Protoc. 2022 Mar 18. 3(1): 101125
    A protocol for investigating lipidomic dysregulation and discovering lipid biomarkers from human serums.
    Moran Chen, Yanhong Hao, Suming Chen.
      Lipids play important roles in various human diseases. Disease-associated lipid dysregulation and biomarkers could provide molecular clues for diagnosis, pathogenesis, and therapy. This protocol provides a step-by-step workflow to investigate lipid dysregulation and discover biomarkers in human serum samples by liquid chromatography-mass spectrometry (LC-MS)-based lipidomics and machine learning analysis. The workflow includes project design, serum collection, sample preparation, data acquisition, data processing, and machine learning analysis. For complete details on the use and execution of this profile, please refer to Hao et al. (2021).
    Keywords:  Bioinformatics; Biotechnology and bioengineering; Clinical Protocol; Health Sciences; Mass Spectrometry; Metabolism
    DOI:  https://doi.org/10.1016/j.xpro.2022.101125
  5. Nat Commun. 2022 Feb 10. 13(1): 782
    Comparative metabolomics with Metaboseek reveals functions of a conserved fat metabolism pathway in C. elegans.
    Maximilian J Helf, Bennett W Fox, Alexander B Artyukhin, Ying K Zhang, Frank C Schroeder.
      Untargeted metabolomics via high-resolution mass spectrometry can reveal more than 100,000 molecular features in a single sample, many of which may represent unidentified metabolites, posing significant challenges to data analysis. We here introduce Metaboseek, an open-source analysis platform designed for untargeted comparative metabolomics and demonstrate its utility by uncovering biosynthetic functions of a conserved fat metabolism pathway, α-oxidation, using C. elegans as a model. Metaboseek integrates modules for molecular feature detection, statistics, molecular formula prediction, and fragmentation analysis, which uncovers more than 200 previously uncharacterized α-oxidation-dependent metabolites in an untargeted comparison of wildtype and α-oxidation-defective hacl-1 mutants. The identified metabolites support the predicted enzymatic function of HACL-1 and reveal that α-oxidation participates in metabolism of endogenous β-methyl-branched fatty acids and food-derived cyclopropane lipids. Our results showcase compound discovery and feature annotation at scale via untargeted comparative metabolomics applied to a conserved primary metabolic pathway and suggest a model for the metabolism of cyclopropane lipids.
    DOI:  https://doi.org/10.1038/s41467-022-28391-9
  6. J Chromatogr A. 2022 Jan 31. pii: S0021-9673(22)00065-6. [Epub ahead of print]1666 462867
    MultiConditionRT: Predicting liquid chromatography retention time for emerging contaminants for a wide range of eluent compositions and stationary phases.
    Amina Souihi, Miklos Peter Mohai, Emma Palm, Louise Malm, Anneli Kruve.
      Structural elucidation of compounds detected with liquid chromatography coupled to high resolution mass spectrometry is a challenging and time-consuming step in the workflow of non-targeted analysis and often requires manual validation of the results. Retention time, alongside exact mass, isotope pattern, fragmentation spectra, and collision cross-section, is valuable information for ruling out unlikely structures and increasing the confidence in others. Different approaches to predict retention times have been used previously for reversed phase chromatography and hydrophilic interaction liquid chromatography (HILIC), but application is limited to a small set of mobile phases and gradient profiles. Here, we expand the toolbox available for retention time predictions by developing a random forest regression model for predicting retention times for four column types and twenty mobile phase systems. MultiConditionRT was built using a dataset containing 78 compounds analyzed with C18 reversed phase, mixed mode, HILIC, and biphenyl columns. In addition, different eluent compositions were used: both methanol and acetonitrile were combined with different aqueous phases with pH from 2.1 to 10.0 (formic acid, acetic acid, trifluoroacetic acid, formate, acetate, bicarbonate, and ammonia). The root mean square error (RMSE) of the test set predictions was 1.55 min for C18 reversed phase, 1.79 min for mixed-mode, 1.93 min for HILIC, and 1.56 min for biphenyl column. Additionally, MultiConditionRT can be applied to different gradient profiles with a general additive model-based calibration approach. The approach of MultiConditionRT was validated externally and internally with 356 and 151 compounds respectively, yielding an RMSE of 2.68 and 2.32 min. 324 and 84 of these compounds were not in the dataset used in the model development.
    Keywords:  Gradient elution; High resolution mass spectrometry; Quantitative structure-retention relationship model; Random forest regression
    DOI:  https://doi.org/10.1016/j.chroma.2022.462867
  7. Mol Nutr Food Res. 2022 Feb 10. e2100985
    A Practitioner's Dilemma Mass Spectrometry-Based Annotation and Identification of Human Plasma and Urinary Polyphenol Metabolites.
    Nikolai Kuhnert, Michael N Clifford.
      The practitioner's dilemma in metabolite assignment can be described as follows: For compound and metabolite identification, should we follow strict guidelines using authentic standards only, or should we accept uncertainties in structure assignment of compounds with the certainty of consequential errors. These uncertainties arise due to limitation of software and databases in combination with the complexity of the human body fluid samples. This article is protected by copyright. All rights reserved.
    Keywords:  Compound annotation; data science; mass spectrometry; metabolites; polyphenols
    DOI:  https://doi.org/10.1002/mnfr.202100985
  8. Talanta. 2022 Jan 26. pii: S0039-9140(22)00060-1. [Epub ahead of print]241 123264
    Biological matrix compatible porous thin film for quick extraction of drugs of abuse from urine prior to liquid chromatography-mass spectrometry analysis.
    Ali Azizi, Fereshteh Shahhoseini, Christina S Bottaro.
      An efficient analytical method is developed using a porous sorptive polymer for thin film microextraction (PSP-TFME) of 8 model drugs from human urine samples. The analysis is conducted with ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The composition of the porous extraction phase, prepared on a stainless steel substrate, has been optimized for basic drugs of abuse and comprises methacrylic acid (MMA) as the monomer and ethylene glycol methacrylate (EGDMA) as the crosslinker. Factors influencing the analyte recovery and method precision (i.e., sample agitation, pH, extraction and washing time, desorption solvent and time) were assessed. The optimized method includes 5 min of direct immersion of the device into the sample, followed by a quick wash in water (1 min) to remove matrix interferences, and then 5 min in acidified methanol for analyte desorption. The extraction devices demonstrated acceptable inter-device variability (2.9-9.3 %RSD for 8 analytes and n = 10 devices) and no detectable difference between batches of devices (p > 0.05 for a 2-sample t-test). The analytical method was linear over a pharmacologically relevant range for each drug (i.e., 0.05-100 ng mL-1 for MDMA and methadone and 2.5-500 ng mL-1 for morphine with R2 varied from 0.9960 to 0.9996). A matrix effect study showed the devices have a high tolerance for complex variable biological matrices. The method also demonstrated excellent data accuracy in the range of 85.3-117.2% for intra-day assays and 88.8-117.9% for inter-day assays. The precision of the method was acceptable and in the range of 0.9-18.6% for intra- and 2.8-16.4% for inter-day assays, respectively.
    Keywords:  Drugs of abuse; High throughput; Liquid chromatography-mass spectrometry; Porous polymers; Sample preparation; Thin film microextraction
    DOI:  https://doi.org/10.1016/j.talanta.2022.123264
  9. Anal Chem. 2022 Feb 09.
    Simultaneous Determination of Vitamin D and Its Hydroxylated and Esterified Metabolites by Ultrahigh-Performance Supercritical Fluid Chromatography-Tandem Mass Spectrometry.
    Bárbara Socas-Rodríguez, Veronika Pilařová, Margareta Sandahl, Cecilia Holm, Charlotta Turner.
      In this study, an analytical method has been developed that, for the first time, allows simultaneous determination of vitamin D2 and vitamin D3 along with their hydroxylated and esterified forms. A group of 12 vitamin D analogues including vitamin D2 and vitamin D3, seven hydroxylated metabolites, and three ester forms were separated in a single 8.0 min run using ultrahigh-performance supercritical fluid chromatography coupled with triple quadrupole tandem mass spectrometry. Electrospray ionization and atmospheric pressure chemical ionization were investigated as ion sources, of which the latter showed a higher ionization efficiency. Chromatographic conditions were thoroughly evaluated by a step-by-step method, whereas an experimental design was applied for the optimization of the ionization parameters. Calibration and repeatability studies were carried out to validate the instrumental methodology showing determination coefficients higher than 0.9992 and good intra- and interday precision with relative standard deviations for areas and retention times lower than 10 and 2.1%, respectively, for all target analytes. Limits of quantification were below 3.03 μg/L for all compounds. The methodology was then validated and applied for the evaluation of human plasma samples in order to demonstrate its applicability to the analysis of vitamin D analogues in biological samples. Samples of five individuals were analyzed. Results show that linoleate-D3, vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 24,25-dihydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 could be detected in most samples, while the two latter also were quantified in all analyzed samples.
    DOI:  https://doi.org/10.1021/acs.analchem.1c04016
  10. J Pharm Biomed Anal. 2022 Jan 29. pii: S0731-7085(22)00038-3. [Epub ahead of print]211 114617
    A selective and accurate liquid chromatography-tandem mass spectrometry method for the quantitation of the novel 5-HT4 receptor partial agonist SUVN-D4010 (Usmarapride) in human plasma and urine.
    Ramakrishna Nirogi, Ilayaraja Kalaikadhiban, Naga Surya Prakash Padala, Hanumanth Rao Pantangi, Prathyusha Chunduru, Abdul Rasheed Mohammed.
      Liquid chromatography and the tandem mass spectrometry method to quantitate SUVN-D4010 (Usmarapride) in human plasma and urine have been developed and fully validated in compliance with regulatory guidelines. The sample preparation technique is simple and rapid consisting of acetonitrile precipitation followed by dilution of supernatant with a compatible solvent. Chromatographic separation was achieved on an X-Bridge C18 (2.1×50 mm, 3.5 µm) column using 0.1% v/v ammonium hydroxide and acetonitrile as mobile phase components, delivered at a flow rate of 0.75 mL min-1. Electrospray Ionization technique in positive mode was used for mass spectrometric detection. Selective reaction monitoring (SRM) transitions of m/z 384.2 → 352.1 for SUVN-D4010 and m/z 388.2 → 356.1 for SUVN-D4010-d4 were used for quantitation. Calibration curves for SUVN-D4010 were linear across the concentration range of 0.3-300 ng mL-1 in human plasma and 5.00-5000 ng mL-1 in human urine. The method generated results with acceptable accuracy (± 9.0%), precision (%CV, ≤8.7), and mean extraction recovery (≥93.4%) with negligible matrix effect in both plasma and urine. SUVN-D4010 was found to be stable in human plasma and urine at the defined storage conditions. The validated method was successfully applied to quantitate SUVN-D4010 in human plasma and urine from a clinical first-in-human study conducted to evaluate its safety, tolerability, and pharmacokinetics in healthy adults.
    Keywords:  5HT(4) receptor partial agonist; Alzheimer’s disease; Human plasma; Incurred sample reproducibility; LC-MS/MS; Pharmacokinetic study; SUVN-D4010
    DOI:  https://doi.org/10.1016/j.jpba.2022.114617
  11. J Pharm Biomed Anal. 2022 Feb 03. pii: S0731-7085(22)00063-2. [Epub ahead of print]212 114642
    Development and application of a highly sensitive LC-MS/MS method for simultaneous quantification of N,N-dimethyltryptamine and two of its metabolites in human plasma.
    Emma Eckernäs, Adam Bendrioua, Chiara Cancellerini, Christopher Timmermann, Robin Carhart-Harris, Kurt-Jürgen Hoffmann, Michael Ashton.
      A highly sensitive LC-MS/MS method for the quantification of N,N-dimethyltryptamine (DMT) and its metabolites indole-3-acetic acid and DMT N-oxide in human plasma has been developed and validated. Chromatography was performed using a diphenyl column with gradient elution (0.1% formic acid in methanol/water). The mass spectrometer was operated in multiple reaction monitoring mode. A methanolic solution containing internal standards 2-methylindole 3-acetic acid and deuterated DMT, was added to plasma samples, followed by protein precipitation with acetonitrile. The samples were centrifuged and supernatants transferred to new tubes and evaporated to dryness before reconstitution in aqueous mobile phase. The method was validated with regards to accuracy, precision, sensitivity, selectivity, recovery, matrix effects, stability, carry-over and dilution integrity. The validated linear range was 0.25-200 nM for DMT and 15-250 nM for DMT N-oxide. For the endogenous compound indole-3-acetic acid a different approach was taken due to its significant presence in blank samples. The change in signal response from a blank sample was used when constructing the calibration curve with linearity demonstrated between elevations of 500-5000 nM above the blank. Applicability of the described method was demonstrated through analysis of plasma samples from healthy volunteers having received intravenous injections of DMT. The presented method for rapid and sensitive quantification of DMT and its metabolites in human plasma can be applied to future studies aiming to characterize DMT disposition and its relationship to immediate psychedelic or long-term antidepressive effects.
    Keywords:  DMT; LC-MS/MS; Metabolites; N,N-dimethyltryptamine; Pharmacokinetics; Psychedelic
    DOI:  https://doi.org/10.1016/j.jpba.2022.114642
  12. J Pharm Biomed Anal. 2021 Dec 30. pii: S0731-7085(21)00673-7. [Epub ahead of print]211 114562
    Method development and validation for simultaneous determination of six tyrosine kinase inhibitors and two active metabolites in human plasma/serum using UPLC-MS/MS for therapeutic drug monitoring.
    Menghua Zhang, Xiaoxue Liu, Zhiyao Chen, Shenhao Jiang, Lin Wang, Min Tao, Liyan Miao.
      Over the past decades, therapeutic drug monitoring (TDM) of tyrosine kinase inhibitors (TKIs) and their main active metabolites has shown benefits in improving treatment efficacy and safety. Therefore, a sensitive, simple and economical ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of six oral tyrosine kinase inhibitors (TKIs) and two active metabolites, including imatinib (IMA), N-desmethyl imatinib (NDIMA), sunitinib (SUNI), N-desethyl sunitinib (NDSUNI), regorafenib (REGO), nilotinib (NILO), dasatinib (DASA) and osimertinib (OSI) in human plasma/serum for therapeutic drug monitoring. The plasma/serum samples were deproteinated with acetonitrile after spiking with two deuterated internal standards (ISs, [2H8]-imatinib and [2H10]-sunitinib) and separated on a 40 °C ACQUITY UPLC® BEH C18 column (1.7 µm, 2.1 mm × 50 mm). The mobile phase was composed of acetonitrile (solution A) and water-formic acid-ammonium acetate (1 M) (994:1:5, v/v/v, solution B). Gradient elution was applied at a flow rate of 0.4 mL/min. Detection was carried out using a Triple Quad 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source in positive mode. The method was validated over the calibration curve (CV) range of 2-400 ng/mL for NDSUNI and DASA, 2.5-500 ng/mL for SUNI, 10-2000 ng/mL NDIMA and OSI, 20-4000 ng/mL for NILO, 30-6000 ng/mL for REGO and 50-10000 ng/mL for IMA using linear regression and 1/x2 weighting. No difference was observed in the matrix effect (ME) among blank human plasma, hemolytic plasma, lipemic plasma, plasma spiked with ten commonly used drugs by cancer patients, or serum samples in general. The standard curve fitted by two standard curves of serum and plasma showed good linearity, and the precision and accuracy results of QC samples in both plasma and serum were acceptable and the concentration was comparable. To provide a clinical reference for the operation, the stability of the whole process from sample collection to drug detection was verified. SUNI and NDSUNI showed obvious photoisomerization under light exposure. Therefore, strict light protection was applied for all sample collection and handling steps of SUNI and NDSUNI. Compared with heparin anticoagulant tubes, the stability of the eight compounds in both whole blood and plasma was better in K3-EDTA and sodium citrate anticoagulant tubes. Given that all the analytes were stable in plasma at 4 °C for 48 h and in whole blood at room temperature for 48 h but OSI and REGO were unstable in whole blood and plasma at room temperature, samples should be centrifuged as soon as possible to be preserved as plasma at 4 °C when OSI or REGO is detected. In conclusion, this validated method can provide support for clinical practice, such as therapeutic drug monitoring (TDM) and pharmacokinetic studies of these six TKIs and two active metabolites.
    Keywords:  LC-MS/MS; Therapeutic drug monitoring; Tyrosine kinase inhibitors
    DOI:  https://doi.org/10.1016/j.jpba.2021.114562
  13. Anal Chem. 2022 Feb 11.
    Noise-Reduced Quantitative Fluorine NMR Spectroscopy Reveals the Presence of Additional Per- and Polyfluorinated Alkyl Substances in Environmental and Biological Samples When Compared with Routine Mass Spectrometry Methods.
    Jeremy R Gauthier, Scott A Mabury.
      Per- and polyfluorinated alkyl substances (PFAS) are ubiquitous throughout the environment. Analysis of PFAS is commonly performed using both targeted and nontargeted mass spectrometry methods. However, it has been demonstrated that measurements of fluorinated compounds in the environment by mass spectrometry often fall short of the total fluorine concentration. In the present study, we employ a 19F NMR technique, which is capable of detailing fluorinated compounds in a sample while providing both quantitative and structural information. Inclusion of a noise-reduction strategy involving the acquisition of arrays of spectra with an increasing number of transients addresses the sensitivity challenges of environmental nuclear magnetic resonance (NMR), improving signal to noise. When this technique is applied to environmental and biological samples including rainwater, lake water, wastewater effluent, serum, and urine, the presence of PFAS, which may have been missed by routine mass spectrometric methods, is revealed. Important resonances in the 19F NMR spectrum such as that of trifluoroacetic acid are brought above the limit of quantification in all samples, allowing detection limits as low as 389 pg/L in rainwater. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which was used to analyze 47 PFAS compounds, accounts for only 3.7-27% of the total fluorine concentration as determined by the NMR strategy in the present study.
    DOI:  https://doi.org/10.1021/acs.analchem.1c05107
  14. Food Chem. 2022 Jan 30. pii: S0308-8146(22)00251-5. [Epub ahead of print]381 132290
    A method for a comprehensive lipidomic analysis of flaxseed (Linum usitatissimum) with the use of LC-Q-TOF-MS and dispersive micro-solid-phase (μDSPE) extraction.
    Inal Bakhytkyzy, Weronika Hewelt-Belka, Agata Kot-Wasik.
      Flaxseed (FS) is one of the richest sources of α-linolenic acid oil and lignans, and it is suggested that the consumption of flaxseed may contribute to the prevention of certain chronic diseases such as many types of cancer, diabetes, cardiovascular diseases and cerebrovascular stroke. Here, we demonstrate a new method for comprehensive FS lipidome profiling with the use of LC-Q-TOF-MS and dispersive micro-solid-phase extraction. The effects of stationary phase amount, flaxseed amount and different organic solvents for non-polar lipid elution on the FS lipidome coverage were investigated. The developed and validated protocol allowed for improved monitoring of both polar and non-polar lipids simultaneously, overcoming the challenge of low- and high-abundance lipid species. Furthermore, the method was applied to characterize a set of brown flaxseed and yellow flaxseed samples, as well as flaxseed meal.
    Keywords:  Dispersive micro-solid-phase extraction; Flaxseed lipidomics; Flaxseed lipids; LC–MS; Lipid extraction; Lipidomics
    DOI:  https://doi.org/10.1016/j.foodchem.2022.132290
  15. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 30. pii: S1570-0232(22)00038-1. [Epub ahead of print]1192 123134
    Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for erythrocyte phosphatidylethanol revealing critical considerations for its use as a clinical biomarker.
    Daniel White, Sean O'Halloran, Sam Salman, Gerry MacQuillan, David A Joyce.
      Erythrocyte membrane-incorporated phosphatidylethanol (PEth) forms only in the presence of ethanol and, once formed, provides a persisting marker for historical alcohol consumption. Relationships between PEth concentration, extent of consumption and time from consumption are under investigation. Threshold values of PEth have been proposed as indicators for any, or for harmful alcohol consumption. Here, we describe an assay for erythrocyte PEth 16:0/18:1 that offers the efficiency needed for routine clinical deployment, in the context of a fully validated methodology. However, we observe that conventional procedures for validating assay methodology are insufficient where the analyte of interest, membrane-incorporated PEth 16:0/18:1, has different physicochemical properties to the soluble PEth 16:0/18:1 and PEth 16:0/18:1-d5 that are used for making calibrator, controls and internal standards. Whereas the internal standard did fully correct for differences in matrix effects and recovery when different extraction solvents were applied to calibrators and controls (in soluble form), it failed to correct for a 1.5-fold difference in the relative efficiency of two solvents, in this case, acetonitrile and isopropanol in extracting PEth from erythrocyte membrane in clinical samples. Differences in the efficiency of the extraction of membrane-bound PEth translate to different results from the same specimen. That can mean that threshold values derived by one methodology cannot be safely generalised to another. That hampers the generalisability of individual laboratory's experience with PEth assay results. Harmonising extraction methodology between laboratories becomes very important where membrane-incorporated PEth itself remains unavailable as an assay standard.
    Keywords:  Erythrocytes; Ethanol; LC-MS/MS; Matrix Effects; Phosphatidylethanol 16:0/18:1; Recovery
    DOI:  https://doi.org/10.1016/j.jchromb.2022.123134
  16. J Vis Exp. 2022 Jan 20.
    Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments.
    Simon Ollivier, Mathieu Fanuel, Hélène Rogniaux, David Ropartz.
      Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.
    DOI:  https://doi.org/10.3791/63451
  17. Electrophoresis. 2022 Feb 07.
    Development of a new Ultra-High-Performance liquid Chromatography-Tandem mass spectrometry (UHPLC-MS/MS) method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay.
    Marco Ballotari, Francesco Taus, Giulia Tolle, Elisa Danese, Romolo M Dorizzi, Franco Tagliaro, Rossella Gottardo.
      Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible of intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC-MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision and recovery. A simple liquid-liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC-MS/MS method vs. the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances. This article is protected by copyright. All rights reserved.
    Keywords:  Digitoxin; Digoxin; Immunoassay; LC-MS/MS
    DOI:  https://doi.org/10.1002/elps.202100290
  18. Crit Rev Anal Chem. 2022 Feb 09. 1-16
    Advancement in Analytical Strategies for Quantification of Biomarkers with a Special Emphasis on Surrogate Approaches.
    Sonam Mishra, Niraj Rajput, Tarang Jadav, Amit Kumar Sahu, Rakesh K Tekade, Pinaki Sengupta.
      Accurate quantification of biomarkers has always been a challenge for many bioanalytical scientists due to their endogenous nature and low concentration in biological matrices. Different analytical approaches have been developed for quantifying biomarkers including enzyme-linked immunosorbent assay, immunohistochemistry, western blotting, and chromatographic techniques assisted with mass spectrometry. Liquid chromatography-tandem mass spectrometry-based quantification of biomarkers has gained more attention over other traditional techniques due to its higher sensitivity and selectivity. However, the primary challenge lies with this technique includes the unavailability of a blank matrix for method development. To overcome this challenge, different analytical approaches are being developed including surrogate analyte and surrogate matrix approach. Such approaches include quantification of biomarkers in a surrogate matrix or quantification of an isotopically labeled surrogate analyte in an authentic matrix. To demonstrate the authenticity of the surrogate approach, it is mandatory to establish quantitative parallelism through validation employing respective surrogate analytes and surrogate matrices. In this review, different bioanalytical approaches for biomarker quantification and recent advancements in the field aiming for improvement in the specificity of the techniques have been discussed. Liquid chromatography-tandem mass spectrometry-based surrogate approaches for biomarker quantification and significance of parallelism establishment in both surrogate matrix and surrogate analyte-based approaches have been critically discussed. In addition, different methods for demonstrating parallelism in the surrogate method have been explained.
    Keywords:  Biomarker quantification; LC–MS/MS; parallelism; surrogate matrix and analyte
  19. Bioinformatics. 2022 Feb 04. pii: btac059. [Epub ahead of print]
    Metaboprep: an R package for pre-analysis data description and processing.
    David A Hughes, Kurt Taylor, Nancy McBride, Matthew A Lee, Dan Mason, Deborah A Lawlor, Nicholas J Timpson, Laura J Corbin.
       MOTIVATION: Metabolomics is an increasingly common part of health research and there is need for pre-analytical data processing. Researchers typically need to characterise the data and to exclude errors within the context of the intended analysis. While some pre-processing steps are common, there is currently a lack of standardization and reporting transparency for these procedures.
    RESULTS: Here we introduce metaboprep, a standardised data processing workflow to extract and characterise high quality metabolomics data sets. The package extracts data from pre-formed worksheets, provides summary statistics and enables the user to select samples and metabolites for their analysis based on a set of quality metrics. A report summarising quality metrics and the influence of available batch variables on the data is generated for the purpose of open disclosure. Where possible, we provide users flexibility in defining their own selection thresholds.
    AVAILABILITY AND IMPLEMENTATION: metaboprep is an open-source R package available at https://github.com/MRCIEU/metaboprep.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btac059
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