bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒01‒16
twenty-five papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Anal Chem. 2022 Jan 13.
      Liquid chromatography-mass spectrometry (LC-MS) is the main workhorse of metabolomics owing to its high degree of analytical sensitivity and specificity when measuring diverse chemistry in complex biological samples. LC-MS-based metabolic profiling of human urine, a biofluid of primary interest for clinical and biobank studies, is not widely considered to be compromised by the presence of endogenous interferences and is often accomplished using a simple "dilute-and-shoot" approach. Yet, it is our experience that broad obscuring signals are routinely observed in LC-MS metabolic profiles and represent interferences that lack consideration in the relevant metabolomics literature. In this work, we chromatographically isolated the interfering metabolites from human urine and unambiguously identified them via de novo structure elucidation as two separate proline-containing dipeptides: N,N,N-trimethyl-l-alanine-l-proline betaine (l,l-TMAP) and N,N-dimethyl-l-proline-l-proline betaine (l,l-DMPP), the latter reported here for the first time. Offline LC-MS/MS, magnetic resonance mass spectrometry (MRMS), and nuclear magnetic resonance (NMR) spectroscopy were essential components of this workflow for the full chemical and spectroscopic characterization of these metabolites and for establishing the coexistence of cis and trans isomers of both dipeptides in solution. Analysis of these definitive structures highlighted intramolecular ionic interactions as responsible for slow interconversion between these isomeric forms resulting in their unusually broad elution profiles. Proposed mitigation strategies, aimed at increasing the quality of LC-MS-based urine metabolomics data, include modification of column temperature and mobile-phase pH to reduce the chromatographic footprint of these dipeptides, thereby reducing their interfering effect on the underlying metabolic profiles. Alternatively, sample dilution and internal standardization methods may be employed to reduce or account for the observed effects of ionization suppression on the metabolic profile.
  2. Anal Chem. 2022 Jan 10.
      Chemical derivatization, especially silylation, is widely used in gas chromatography coupled to mass spectrometry (GC-MS). By introducing the trimethylsilyl (TMS) group to substitute active hydrogens in the molecule, thermostable volatile compounds are created that can be easily analyzed. While large GC-MS libraries are available, the number of spectra for TMS-derivatized compounds is comparatively small. In addition, many metabolites cannot be purchased to produce authentic library spectra. Therefore, computationally generated in silico mass spectral databases need to take TMS derivatizations into account for metabolomics. The quantum chemistry method QCEIMS is an automatic method to generate electron ionization (EI) mass spectra directly from compound structures. To evaluate the performance of the QCEIMS method for TMS-derivatized compounds, we chose 816 trimethylsilyl derivatives of organic acids, alcohols, amides, amines, and thiols to compare in silico-generated spectra against the experimental EI mass spectra from the NIST17 library. Overall, in silico spectra showed a weighted dot score similarity (1000 is maximum) of 635 compared to the NIST17 experimental spectra. Aromatic compounds yielded a better prediction accuracy with an average similarity score of 808, while oxygen-containing molecules showed lower accuracy with only an average score of 609. Such similarity scores are useful for annotation of small molecules in untargeted GC-MS-based metabolomics, suggesting that QCEIMS methods can be extended to compounds that are not present in experimental databases. Despite this overall success, 37% of all experimentally observed ions were not found in QCEIMS predictions. We investigated QCEIMS trajectories in detail and found missed fragmentations in specific rearrangement reactions. Such findings open the way forward for future improvements to the QCEIMS software.
  3. Chirality. 2022 Jan 14.
      Branched-chain fatty acids (BCFAs) are mostly saturated fatty acids with one or more methyl, seldom ethyl, branches in the alkyl chain. They are derived from branched-chain amino acids, ruminant-derived food, or biosynthetic side products of acetyl-CoA carboxylase. They possess iso- (branching at penultimate carbon) and anteiso-fatty acid structure (branching at antepenultimate carbon) or are branched at any other position of the carbon chain. Except for iso-fatty acids, BCFAs are chiral. They are commonly analyzed by GC-MS, while there is a lack of enantioselective LC-MS methods. In this work, we present a methodology for targeted enantioselective UHPLC-ESI-MS/MS metabolomics of BCFAs. It makes use of precolumn derivatization with 1-naphthylamine and reversed-phase elution conditions. A homologous series of short BCFA analytes with distinct chain lengths (having up to eight carbon atoms), branching type (methyl or ethyl), and position of branching (2, 3, and 4, anteiso and iso) has been systematically studied on six commercially available polysaccharide UHPLC columns. Chiralpak IB-U exhibited the highest and broadest enantioselectivity while IH-U maintained enantioselectivity also for BCFAs with chirality distant from the carboxylic function (i.e., with other branching than in 2-position). The method was used to assign the absolute configuration of a 4-methylhexanoic acid side chain of a natural product from Streptomyces sp. SHP 22-7. The potential of the corresponding UHPLC-ESI-QTOF-MS/MS assay for analyzing stereoselectively BCFAs and other short organic acids by untargeted analysis in human urine was further elucidated in a preliminary proof-of-principle test.
    Keywords:  bacterial fatty acids; chiral separation; lipidomics; natural products; organic acids; polysaccharide CSPs
  4. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 06. pii: S1570-0232(22)00003-4. [Epub ahead of print]1189 123099
      We evaluated the performance of three different single-phase extraction methods to be used before untargeted lipidomics analysis by Liquid Chromatography High-Resolution Mass Spectrometry. Lipids were extracted from a pool of healthy human donors' plasma in triplicates and run in both positive and negative ESI. The most satisfactory results were attained using methanol/chloroform (2:1, v/v) mixture. Eventually, we evaluated whether a filtration of the samples could be beneficial to yield cleaner and more mass-friendly extracts. Instead of using syringes, we set up a method we called tip-tip filtration, which requires the usage of a filtrating pipette tip. This way of purification led to superior results than the solvent extraction method alone. This additional procedure not only increased reproducibility but also allowed the same lipid coverage. In addition, it permitted to spare time and money, as tip-tip filtration is not particularly expensive nor time-consuming and hopefully it may be useful to increase analytical column lifetime.
    Keywords:  Lipid extraction; Mass spectrometry; Sample pre-treatment; Untargeted lipidomics
  5. Molecules. 2022 Jan 03. pii: 283. [Epub ahead of print]27(1):
      Vicinal diols are important signaling metabolites of various inflammatory diseases, and some of them are potential biomarkers for some diseases. Utilizing the rapid reaction between diol and 6-bromo-3-pyridinylboronic acid (BPBA), a selective and sensitive approach was established to profile these vicinal diols using liquid chromatography-post column derivatization coupled with double precursor ion scan-mass spectrometry (LC-PCD-DPIS-MS). After derivatization, all BPBA-vicinal-diol esters gave a pair of characteristic isotope ions resulting from 79Br and 81Br. The unique isotope pattern generated two characteristic fragment ions of m/z 200 and 202. Compared to a traditional offline derivatization technique, the new LC-PCD-DPIS-MS method retained the capacity of LC separation. In addition, it is more sensitive and selective than a full scan MS method. As an application, an in vitro study of the metabolism of epoxy fatty acids by human soluble epoxide hydrolase was tested. These vicinal-diol metabolites of individual regioisomers from different types of polyunsaturated fatty acids were easily identified. The limit of detection (LOD) reached as low as 25 nM. The newly developed LC-PCD-DPIS-MS method shows significant advantages in improving the selectivity and therefore can be employed as a powerful tool for profiling vicinal-diol compounds from biological matrices.
    Keywords:  double precursor ion scan; epoxide hydrolase; online post-column derivatization; vicinal diols
  6. Anal Chem. 2022 Jan 13.
      Standardization is essential in lipidomics and part of a huge community effort. However, with the still ongoing lack of reference materials, benchmarking quantification is hampered. Here, we propose traceable lipid class quantification as an important layer for the validation of quantitative lipidomics workflows. 31P nuclear magnetic resonance (NMR) and inductively coupled plasma (ICP)-mass spectrometry (MS) can use certified species-unspecific standards to validate shotgun or liquid chromatography (LC)-MS-based lipidomics approaches. We further introduce a novel lipid class quantification strategy based on lipid class separation and mass spectrometry using an all ion fragmentation (AIF) approach. Class-specific fragments, measured over a mass range typical for the lipid classes, are integrated to assess the lipid class concentration. The concept proved particularly interesting as low absolute limits of detection in the fmol range were achieved and LC-MS platforms are widely used in the field of lipidomics, while the accessibility of NMR and ICP-MS is limited. Using completely independent calibration strategies, the introduced validation scheme comprised the quantitative assessment of the complete phospholipid sub-ome, next to the individual lipid classes. Komagataella phaffii served as a prime example, showcasing mass balances and supporting the value of benchmarks for quantification at the lipid species level.
  7. J Mass Spectrom Adv Clin Lab. 2022 Jan;23 44-49
      Introduction: We have developed a set of tools built with open-source software that includes both a database and a visualization component to collect LC-MS/MS data and monitor quality control parameters.Description of tool: To display LC-MS/MS data we built a parsing tool using Python and standard libraries to parse the XML files after each clinical run. The tool parses the necessary information to store a database comprised of three distinct tables. Another component to this toolkit is an interactive data visualization tool that uses the data from the database. There are 5 different visualizations that present the data based on interchangeable parameters.
    Evaluation of tool: Using histogram visualization, we assessed how quality control parameters that feed our quality control algorithm, SMACK, which assists to improve the efficiency of data review and results, performed against the collective data. Using the newly identified QC parameter values from the toolkit, we compared the output of the SMACK algorithm; the number of QC flags changed in that there was a 1.7% (31/1944 observations) increase in flags and a 7.1% (138/1944 observations) decrease in presumed false positive flags, increasing the overall performance of SMACK which helped staff focus their time on reviewing more concerning QC failures.
    Discussion: We have developed a customizable web-based dashboard for instrument performance monitoring for our opiate confirmation LC-MS/MS assay using data collected with each batch. The web-based platform allows users to monitor instrument performance and can encompass other instruments throughout the laboratory. This information can help the laboratory take proactive measures to maintain instruments, ultimately reducing the amount down time needed for maintenance.
    Keywords:  Dashboard; Database; GB, Gigabyte; LC-MS/MS, Liquid chromatography tandem mass spectrometry; LLOQ, Lower limit of quantification; MB, Megabyte; Mass spectrometry; Python; QC, Quality control; Quality control; RRT, Relative retention time; Visualization
  8. J Anal Toxicol. 2022 Jan 12. pii: bkac004. [Epub ahead of print]
      This manuscript reported a fast and rapid qualitative screening method for abuse drugs in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS). The scope of the abuse drugs under investigation included methamphetamine (MA), amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), paramethoxymethamphetamine (PMMA), ephedrine, pseudoephedrine, ketamine (KET), deschloroketamine (DCK), 2-fluorodeschloroketamine (2-F-DCK) and deschloro-N-ethylketamine (2-oxo-PCE). The method employed a dual mode extraction (DME) column as a novel clean up method for the urine matrix. To an aliquot of 0.2 mL urine, internal standards (ISTDs) and 0.4 mL of acidified methanol were added. After vortex and centrifugation, the supernatant was passed through a dual mode extraction (DME) column before LC-MS/MS analysis. Chromatographic separation was achieved with a C18 column by gradient elution. The limits of detection (LODs) for MA, AMP, MDMA, MDA and PMMA were 3 ng/mL; whereas those for ephedrine and pseudoephedrine were 10 ng/mL and those for KET, DCK, 2-F-DCK and 2-oxo-PCE were 1 ng/mL. The matrix effects ranged from -12% to 7% (%CV from 4% to 19%). This method is fit for the intended purpose for forensic toxicology, as well as for forensic analysis of drugs facilitating sexual assault (DFSA) and other criminal acts.
    Keywords:  DME; LC-MS/MS; abuse drugs; amphetamine; ketamine; screening; toxicology; urine
  9. J Mass Spectrom. 2021 Dec 26. 57(2): e4804
      Polycyclic aromatic hydrocarbons (PAHs) are common pollutants present in atmospheric aerosols and other environmental mixtures. They are of particular air quality and human health concerns as many of them are carcinogenic toxins. They also affect absorption of solar radiation by aerosols, therefore contributing to the radiative forcing of climate. For environmental chemistry studies, it is advantageous to quantify PAH components using the same analytical technics that are commonly applied to characterize a broad range of polar analytes present in the same environmental mixtures. Liquid chromatography coupled with photodiode array and high-resolution mass spectrometric detection (LC-PDA-HRMS) is a method of choice for comprehensive characterization of chemical composition and quantification of light absorption properties of individual organic compounds present in the environmental samples. However, quantification of non-polar PAHs by this method is poorly established because of their imperfect ionization in electrospray ionization (ESI) technique. This tutorial article provides a comprehensive evaluation of the quantitative analysis of 16 priority pollutant PAHs in a standard reference material using the LC-MS platform coupled with the ESI source. Results are further corroborated by the quantitation experiments using an atmospheric pressure photoionization (APPI) method, which is more sensitive for the PAH detection. The basic concepts and step-by-step practical guidance for the PAHs quantitative characterization are offered based on the systematic experiments, which include (1) Evaluation effects of different acidification levels by formic acid on the (+)ESI-MS detection of PAHs. (2) Comparison of detection limits in ESI+ versus APPI+ experiments. (3) Investigation of the PAH fragmentation patterns in MS2 experiments at different collision energies. (4) Calculation of wavelength dependent mass absorption coefficient (MACλ ) of the standard mixture and its individual PAHs using LC-PDA data. (5) Assessment of the minimal injected mass required for accurate quantification of MACλ of the standard mixture and of a multi-component environmental sample.
    Keywords:  analysis of complex mixtures; atmospheric pressure photoionization; electrospray ionization; high performance liquid chromatography; mass spectrometry; photodiode array detector; polycyclic aromatic hydrocarbons; quantitation
  10. Clin Chim Acta. 2022 Jan 06. pii: S0009-8981(21)00457-5. [Epub ahead of print]527 1-10
      BACKGROUND AND AIMS: As numerous studies have reported the concentration-exposure relationships of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), therapeutic drug monitoring is a promising approach in lung cancer treatment, aiming to avoid treatment failure or toxicity. A new method for the simultaneous analysis of five EGFR-TKIs (afatinib, erlotinib, gefitinib, icotinib and osimertinib) and their metabolites in human plasma samples was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS).MATERIALS AND METHODS: Afatinib-d6, erlotinib-d6, OSI-420-d4, gefitinib-d6 and osimertinib-C13,d3 were used as internal standards (ISs). The samples were prepared by liquid-liquid extraction using tert-butyl methyl ether. Chromatographic separation was undertaken on an XBridge C18 column using a linear gradient elution. LC-MS/MS was conducted in positive ionization mode with multiple reaction monitoring.
    RESULTS: The proposed method showed satisfactory results in terms of linearity, sensitivity, specificity, precision (intra- and inter-day coefficients of variation ranged from 1.1 to 13.9%), and accuracy (from 93.3 to 111.1%). The IS-normalized matrix factors were below 15%. The sensitivity and linearity were highly appropriate for the expected concentrations according to the analysis of samples from non-small cell lung caner (NSCLC) patients who received EGFR-TKIs.
    CONCLUSIONS: The proposed method showed an acceptable reproducibility, high sensitivity and selectivity, and low matrix effects. This method could be significant for monitoring plasma concentrations of the mentioned EGFR-TKIs in NSCLC patients, aiming to improve the efficacy and safety of targeted therapies.
    Keywords:  EGFR-TKIs; LC–MS/MS; Metabolites; Non-small cell Lung cancer; Therapeutic drug monitoring
  11. J Chromatogr A. 2021 Dec 30. pii: S0021-9673(21)00915-8. [Epub ahead of print]1665 462793
      The objective of this work was to develop a quantitative multi-residue method for analysing antiviral drug residues and their metabolites in poultry meat samples. Antiviral drugs are not licensed for the treatment of influenza in food producing animals. However, there have been some reports indicating their illegal use in poultry. In this study, a method was developed for the analysis of 15 antiviral drug residues in poultry muscle (chicken, duck, quail and turkey) using liquid chromatography coupled to tandem mass spectrometry. This included 13 drugs against influenza and associated metabolites, but also two drugs employed for the treatment of herpes (acyclovir and ganciclovir). The method required the development of a novel chromatographic separation using a hydrophilic interaction chromatographic (HILIC) BEH amide column, which was necessary to retain the highly polar compounds. The analytes were detected using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. A range of different sample preparation protocols suitable for polar compounds were evaluated. The most effective procedure was based on a simple acetonitrile-based protein precipitation step followed by a further dilution in a methanol/water solution. The confirmatory method was validated according to the EU 2021/808 guidelines on different species including chicken, duck, turkey and quail. The validation was performed using various calibration curves ranging from 0.1 µg kg-1to 200 µg kg-1, according to the analyte. Depending on the analyte sensitivity, decision limits achieved ranged from 0.12 µg kg-1 for arbidol to 34.7 µg kg-1 for ribavirin. Overall, the reproducibility precision values ranged from 2.8% to 22.7% and the recoveries from 84% to 127%. The method was applied to 120 commercial poultry samples from the Irish market, which were all found to be residue-free.
    Keywords:  Antiviral drug residues; HILIC; Influenza; LC-MS/MS; Poultry muscle
  12. Anal Chem. 2022 Jan 14.
      The mass spectrometry imaging (MSI) technique is widely used in several fields due to its ability to provide spatial information of samples. However, for existing MSI methods, the sample is typically placed on a two-dimensional (2D) platform and is scanned back and forth. As a result, the platform size limits the imaging size. This paper proposes a new MSI method that involves the initial imprinting of chemicals on a two-dimensional string plane area. The string plane was then unraveled to a one-dimensional (1D) string, and the chemicals imprinted on it were ionized using a lab-made ion source. Finally, a 2D MSI image was reconstructed through data processing (2D-1D-2D mass imaging). Compared with traditional MSI methods, the imaging size is no longer limited by the platform size, making it possible to perform the MSI of large samples. As proof of concept, this method was used to image an intact seedling of Broussonetia papyrifera. As a result, clear and overall MS images were obtained, demonstrating the ability of this method to analyze large samples.
  13. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 10. pii: S1570-0232(21)00575-4. [Epub ahead of print]1190 123094
      Liquid chromatography (LC) - mass spectrometry quantitative analysis of substances in biological samples is usually performed in the multiple reaction monitoring (MRM) variant. In complex biological matrices, strong interferences can be observed when using the LC-MRM method. Interference levels can be significantly reduced by using LC - multiple reaction monitoring cubed (MRM3). 6-sulfatoxymelatonin (6-SM) is a metabolite of melatonin, an important regulator of many biological processes. The quantitative analysis of 6-SM in urine allows monitoring of the melatonin level in the blood. The aim of the present work was to evaluate the LC-MRM3 method for the quantitative determination of 6-SM in urine. We found that for 6-SM in aqueous solutions, under some parameters of the MRM3 experiment, the effect of degradation of the MRM3 signal is observed. When analyzing 6-SM in urine, this signal degradation effect was significantly reduced. We have shown that optimization of such parameters of the MRM3 method as the linear ion trap fill time, the number of scans to sum, and the range of triple-stage scan allows obtaining the LC-MRM3 method, which is comparable to the LC-MRM in sensitivity and significantly exceeds it in selectivity.
    Keywords:  6-sulfatoxymelatonin; Linear ion trap; MRM(3); Multiple reaction monitoring cubed
  14. Metabolomics. 2022 Jan 09. 18(1): 11
      INTRODUCTION: High-throughput screening (HTS) is emerging as an approach to support decision-making in chemical safety assessments. In parallel, in vitro metabolomics is a promising approach that can help accelerate the transition from animal models to high-throughput cell-based models in toxicity testing.OBJECTIVE: In this study we establish and evaluate a high-throughput metabolomics workflow that is compatible with a 96-well HTS platform employing 50,000 hepatocytes of HepaRG per well.
    METHODS: Low biomass cell samples were extracted for metabolomics analyses using a newly established semi-automated protocol, and the intracellular metabolites were analysed using a high-resolution spectral-stitching nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) method that was modified for low sample biomass.
    RESULTS: The method was assessed with respect to sensitivity and repeatability of the entire workflow from cell culturing and sampling to measurement of the metabolic phenotype, demonstrating sufficient sensitivity (> 3000 features in hepatocyte extracts) and intra- and inter-plate repeatability for polar nESI-DIMS assays (median relative standard deviation < 30%). The assays were employed for a proof-of-principle toxicological study with a model toxicant, cadmium chloride, revealing changes in the metabolome across five sampling times in the 48-h exposure period. To allow the option for lipidomics analyses, the solvent system was extended by establishing separate extraction methods for polar metabolites and lipids.
    CONCLUSIONS: Experimental, analytical and informatics workflows reported here met pre-defined criteria in terms of sensitivity, repeatability and ability to detect metabolome changes induced by a toxicant and are ready for application in metabolomics-driven toxicity testing to complement HTS assays.
    Keywords:  Chemical risk assessment; Direct infusion mass spectrometry; HepaRG; High-throughput screening; In vitro metabolomics; Toxicology
  15. J Agric Food Chem. 2022 Jan 13.
      A comprehensive liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach was developed to discriminate honey harvested from Apis mellifera ligustica Spinola (A. mellifera) and Apis cerana cerana Fabricius (A. cerana). Based on an untargeted strategy, ultrahigh-performance liquid chromatography electrospray ionization quadrupole orbitrap high-resolution mass spectrometry (UPLC Q-Orbitrap) was combined with chemometrics techniques to screen and identify tentative markers from A. mellifera and A. cerana honey. In targeted metabolomics analysis, a sensitive method of solid-phase extraction followed by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS) was established for quantifying three markers, and the results showed that 3-amino-2-naphthoic acid and methyl indole-3-acetate could be considered markers of A. cerana honey, as they were present in higher amounts in A. cerana honey than in A. mellifera honey, whereas kynurenic acid was determined to be a marker of A. mellifera honey. This work highlights critical information for the authentication of A. cerana and A. mellifera honey.
    Keywords:  A. mellifera and A. cerana; UPLC Q-Orbitrap; UPLC-MS/MS; honey; untargeted and targeted discrimination
  16. Transl Clin Pharmacol. 2021 Dec;29(4): 216-225
      Trimethylamine N-oxide (TMAO) is a small molecular amine oxide generated from dietary choline and carnitine through intestinal microbial metabolism. Recently, TMAO has attracted much public attention as its role in disease progression has been proven in many clinical studies. The plasma concentration of TMAO in humans was found to be positively associated with the increased risk of many diseases including cardiovascular diseases and chronic kidney diseases. To achieve accurate and sensitive quantitation of TMAO for clinical applications, we established and validated a simple quantitative method using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system. We constructed an eight-point calibration curve in an artificial surrogate matrix instead of the commonly used biological matrices to avoid interference from the endogenous TMAO. The calibration curve showed excellent linearity in the range of 1 to 5,000 ng/mL, with a correlation coefficient (R2) higher than 0.996 in each validation batch. Moreover, both the intra-day and inter-day assays achieved satisfactory precision and accuracy results ranging from 1.65-7.15% and 96.36-111.43%, respectively. Further, this method was cross-validated using a human plasma matrix and applied to a clinical pharmacology study. Overall, these results demonstrate that the developed quantitation method is applicable in clinical research for monitoring disease progression and evaluating drug effects.
    Keywords:  Biomarkers; Liquid Chromatography; TMAO; Tandem Mass Spectrometry
  17. J Mass Spectrom. 2022 Mar;57(3): e4807
      Mass spectrometry is a powerful tool in disparate areas of chemistry, but its characteristic strength of sensitivity can be an Achilles heel when studying highly reactive organometallic compounds. A quantity of material suitable for mass spectrometric analysis often represents a tiny grain or a very dilute solution, and both are highly susceptible to decomposition due to ambient oxygen or moisture. This complexity can be frustrating to chemists and analysts alike: the former being unable to get spectra free of decomposition products and the latter often being poorly equipped to handle reactive samples. Fortunately, many creative solutions to such problems have been developed. This review summarizes some key methods for handling reactive samples in conjunction with the various ionization methods most frequently employed for their analysis.
  18. J Chromatogr A. 2021 Dec 30. pii: S0021-9673(21)00914-6. [Epub ahead of print]1664 462792
      Retention time prediction in high-performance liquid chromatography (HPLC) is the subject of many studies since it can improve the identification of unknown molecules in untargeted profiling using HPLC coupled with high-resolution mass spectrometry. Lots of approaches were developed for retention time prediction in liquid chromatography for a different number of molecules considering various molecular properties and machine learning algorithms. The recently built large retention time data set of standard compounds from the Metabolite and Chemical Entity Database (METLIN) allows researchers to create a model that can be used for retention time prediction of small molecules with wide varieties of structures and physicochemical properties. The ability to predict retention times using the largest data set was studied for different architectures of deep learning models that were trained on molecular fingerprints, and SMILES (string representation of a molecule) represented as one-hot matrices. The best result was achieved with a one-dimensional convolutional neural network (1D CNN) that uses SMILES as an input. The proposed model reached the mean absolute error and the median absolute error equal to 34.7 and 18.7 s, respectively, which outperformed the results previously obtained for this data set. The pre-trained 1D CNN on the METLIN SMRT data set was transferred on five other data sets to evaluate the generalization ability.
    Keywords:  Deep learning; RP-HPLC; Retention time prediction
  19. Foods. 2022 Jan 01. pii: 111. [Epub ahead of print]11(1):
      In this study, matrix solid phase dispersion (MSPD) microextraction combined with supercritical fluid chromatography-ion mobility quadrupole time-of-flight mass spectrometry (SFC/IM-QTOF-MS) was used to analyze the multipolar compounds in plant tea. The parameters of stationary phase, mobile phase, make-up solution, temperature, and back pressure were optimized. The target analytes were gradient eluted in 8 min by supercritical CO2 on a Zorbax RX-SIL column. Collisional Cross Section (CCS) values for single and multiple fields were measured. A series of validation studies were carried out under the optimal conditions, and the linear relationship and reproducibility were good. The limits of detection were 1.4 (Scoparone (1))~70 (Naringenin (4)) ng/mL, and the limits of quantification were 4.7 (Scoparone (1))~241 (Naringenin (4)) ng/mL. The recoveries of most compounds ranged from 60.7% to 127%. As a consequence, the proposed method was used for the separation and quantitative analysis of active ingredients in caulis dendrobii.
    Keywords:  caulis dendrobii; ion mobility quadrupole time-of-flight mass spectrometry; matrix solid phase dispersion; multipolar compounds; plant tea; supercritical fluid chromatography
  20. J Am Soc Mass Spectrom. 2022 Jan 11.
      The increasing popularity of high-resolution mass spectrometry has led to many custom software solutions to process, interpret, and reveal information from high-resolution mass spectra. Although there are numerous software packages for peak-picking, calibration, and formula-finding, there are additional layers of information available when it comes to detecting repeated motifs from polymers or molecules with repeating structures or products of chemical or biochemical transformations that exhibit systematic, serial chemical changes of mass. Constellation is an open-source, Python-based web application that allows the user first to expand their high-resolution mass data into the mass defect space, after which a trend finding algorithm is used for supervised or unsupervised detection of repeating motifs. Many adjustable parameters allow the user to tailor their trend-search to target particular chemical moieties or repeating units, or search for all potential motifs within certain limits. The algorithm has a built-in optimization routine to provide a good starting point for the main trend finding parameters before user customization. Visualization tools allow interrogation of the data and any trends/patterns to a highly specific degree and save publication-quality images directly from the interface. As Constellation is deployed as a web application, it is easily used by anyone with a web browser; no software download or high-powered computer is required, as computations are performed on a remote high-powered data server run by our group.
  21. J Pharm Biomed Anal. 2022 Jan 03. pii: S0731-7085(21)00687-7. [Epub ahead of print]210 114576
      N-nitrosamines (NAs) are common toxic substances that have a strong correlation with many human diseases, such as liver damage and cancer. However, there is a lack of studies on methods involving the detection of NAs in biological samples, possibly owing to the interference of complex biological matrices and the influence of endogenous NAs. In this work, solid-phase extraction with mixed solid phases and adsorption sedimentation were used to successfully establish a gas chromatography-mass spectrometry (GC-MS) method for detecting eight NAs in rat faeces. Chromatographic separation of analytes was performed with Agilent VF-WAXms (30 m × 0.25 mm, 0.25 µm) GC columns. The LLOQs of eight NAs were set to the concentration of 0.5 ng/g and the obtained standard curves were linear, and correlation coefficients (r) were ≥ 0.99 for samples with concentration ranges of 0.5-500 ng/g. The inter and intra-assay precisions were< 15% for all analytes in the quality control samples, and the accuracies ranged from 88.67% to 108.33%. The extraction recoveries were above 78.56% for seven NAs, and a significant matrix effect was not observed. The application of this method revealed that the levels of NAS in the faeces of rats with colorectal cancer were higher than those of normal rats. Additionally, the effect of a high nitrite diet on NAs in faeces was analysed; the results confirmed that a high nitrite diet might contribute to an abnormal increase in NAs. Our work provides an analytical method for further in vivo study of NAs. Furthermore, a pilot study on the relationship between NAs and colorectal cancer was completed.
    Keywords:  Colorectal cancer; GC-MS; Gut microbiota; High nitrite diet; NDMA; Nitrosamines
  22. BMJ Nutr Prev Health. 2021 ;4(2): 374-384
      Background: Metabolomics analysis of human stool samples is of great interest for a broad range of applications in biomedical research including early detection of colorectal neoplasms. However, due to the complexity of metabolites there is no consensus on how to process samples for stool metabolomics measurements to obtain a broad coverage of hydrophilic and hydrophobic substances.Methods: We used frozen stool samples (50 mg) from healthy study participants. Stool samples were processed after thawing using eight different processing protocols and different solvents (solvents such as phosphate-buffered saline, isopropanol, methanol, ethanol, acetonitrile and solvent mixtures with or without following evaporation and concentration steps). Metabolites were measured afterwards using the MxP Quant 500 kit (Biocrates). The best performing protocol was subsequently applied to compare stool samples of participants with different dietary habits.
    Results: In this study, we were able to determine up to 340 metabolites of various chemical classes extracted from stool samples of healthy study participants with eight different protocols. Polar metabolites such as amino acids could be measured with each method while other metabolite classes, particular lipid species (better with isopropanol and ethanol or methanol following a drying step), are more dependent on the solvent or combination of solvents used. Only a small number of triglycerides or acylcarnitines were detected in human faeces. Extraction efficiency was higher for protocols using isopropanol (131 metabolites>limit of detection (LOD)) or those using ethanol or methanol and methyl tert-butyl ether (MTBE) including an evaporation and concentration step (303 and 342 metabolites>LOD, respectively) than for other protocols. We detected significant faecal metabolite differences between vegetarians, semivegetarians and non-vegetarians.
    Conclusion: For the evaluation of metabolites in faecal samples, we found protocols using solvents like isopropanol and those using ethanol or methanol, and MTBE including an evaporation and concentration step to be superior regarding the number of detected metabolites of different chemical classes over others tested in this study.
    Keywords:  dietary patterns
  23. ACS Appl Bio Mater. 2021 Aug 16. 4(8): 6236-6243
      Online coupling in-tube solid-phase microextraction (SPME) with mass spectrometry (MS) was achieved by directly utilizing a monolithic SPME capillary as the electrospray emitter. A porous organic polymer with a highly porous structure can not only provide excellent extraction efficiency due to its high specific surface area but also increase the ionization efficiency and stability by producing smaller electrospray droplets. This online coupling strategy significantly simplifies the device as well as the operation process. The whole analytical process only takes about 12 min with a sample amount of 100 μL. By choosing an ionic liquid-based organic polymer with high extraction ability and good stability and adjusting a suitable porous structure for better electrospray, the developed in-tube SPME-MS method showed excellent extraction performance and good reusability without clogging events. As a proof of concept, the proposed method was applied for the analysis of non-steroid anti-inflammatory drugs in plasma and urine samples. The method showed a wide linear range (0.1-200 ng/mL), good linearity (R2 ≥ 0.99), and good reproducibility (intra-day relative standard deviations (RSDs) <3.00% and inter-day RSDs <6.29%).
    Keywords:  direct mass spectrometry; electrospray emitter; human plasma; human urine; monolithic polymer; non-steroid anti-inflammatory drugs; solid-phase microexraction
  24. Rapid Commun Mass Spectrom. 2022 Jan 10. e9255
      RATIONALE: Foscarnet (FCV) is used to treat cytomegalovirus, human herpesvirus, and human immunodeficiency virus infections. It is a low molecular weight compound containing carboxylate and phosphate groups. There are no reports on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze FCV via bioanalysis. In the present study, we developed the ion-pair LC-MS/MS method to analyze FCV in human serum and cerebrospinal fluid (CSF).METHODS: FCV was extracted from human serum and CSF by weak anion exchange (WAX) solid-phase extraction. The LC-MS/MS systems were coated with 0.1% phosphoric acid in methanol to avoid non-specific absorption. FCV was detected by ion-pair LC-MS/MS with dibutyl ammonium acetate. Multiple reaction monitoring transition of FCV in the negative ion mode was selected at m/z 125.1→62.9.
    RESULTS: FCV was selectively detected in human serum and CSF, and the liner range was 5-1000 μM (R2 = 0.99). The intraday and interday accuracy and precision were within ±15%. FCV was constantly extracted from human serum and CSF by WAX solid phase extraction (recovery ratio = 76.0%-77.9%). No matrix effect was observed.
    CONCLUSIONS: A robust LC-MS/MS method to analyze FCV was successfully developed. FCV was selectively measured by LC-MS/MS in very low extract volumes (20 μL). The method will be useful in evaluating the FCV level in human serum and CSF.
  25. Bioinformatics. 2022 Jan 06. pii: btac003. [Epub ahead of print]
      MOTIVATION: Recent technological developments have facilitated an expansion of microbiome-metabolome studies, in which samples are assayed using both genomic and metabolomic technologies to characterize the abundances of microbial taxa and metabolites. A common goal of these studies is to identify microbial species or genes that contribute to differences in metabolite levels across samples. Previous work indicated that integrating these datasets with reference knowledge on microbial metabolic capacities may enable more precise and confident inference of microbe-metabolite links.RESULTS: We present MIMOSA2, an R package and web application for model-based integrative analysis of microbiome-metabolome datasets. MIMOSA2 uses genomic and metabolic reference databases to construct a community metabolic model based on microbiome data and uses this model to predict differences in metabolite levels across samples. These predictions are compared with metabolomics data to identify putative microbiome-governed metabolites and taxonomic contributors to metabolite variation. MIMOSA2 supports various input data types and customization with user-defined metabolic pathways. We establish MIMOSA2's ability to identify ground truth microbial mechanisms in simulation datasets, compare its results with experimentally inferred mechanisms in honeybee microbiota, and demonstrate its application in two human studies of inflammatory bowel disease. Overall, MIMOSA2 combines reference databases, a validated statistical framework, and a user-friendly interface to facilitate modeling and evaluating relationships between members of the microbiota and their metabolic products.
    AVAILABILITY AND IMPLEMENTATION: MIMOSA2 is implemented in R under the GNU General Public License v3.0 and is freely available as a web server at and as an R package from
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.