bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒04‒11
twelve papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Mar 04. pii: S1570-0232(21)00094-5. [Epub ahead of print]1173 122614
      Pyrithione glucuronide (PTG) and 2-thiopyridine glucuronide (ThPG) have been reported to be the major urinary metabolites in multiple animal species following administration of zinc pyrithione (ZnPT). However, the formation of these metabolites has never been confirmed in humans. A simple and rugged ultra-high-performance liquid chromatography high resolution mass spectrometry (UHPLC-MS/HRMS) method was developed and validated for the quantification of PTG and ThPG to investigate human metabolism of pyrithione following topical application of ZnPT as a shampoo. A UHPLC-MS/HRMS method was required due to the matrix interferences that were observed with the typical industry standard HPLC/tandem mass spectrometry (LC-MS/MS) methodology based on nominal mass triple quadrupole (QQQ) platform approach. Using UPLC-MS/HRMS, both PTG and ThPG were identified in human urine following topical application of ZnPT. The presence of these human urinary metabolites of pyrithione are consistent with findings from earlier studies in multiple animal species and suggest the metabolism of pyrithione is similar amongst those mammalian species studied.
    Keywords:  High resolution mass spectrometry; Pyrithione; Pyrithione glucuronide; Thiopyridine glucuronide; Urinary metabolites
  2. J Sep Sci. 2021 Apr 09.
      The measurement of steroid hormones provided critical information in the clinical evaluation of endocrine disorders. In this study, we developed a high throughput solid-phase extraction method for the analysis of 26 steroids in human serum and plasma samples by liquid chromatography-tandem mass spectrometry. Chromatographic condition and sample preparation were optimized to achieve good separation and maximum sensitivity for these analytes. Under the optimum conditions, good linearities were achieved in the quantitative range for each steroid hormone with the correlation coefficients (r) larger than 0.99. The limits of quantitation of the method were in the range from 0.0005 to 0.7901 ng/mL. The recoveries were in the range of 87.2% to 114.2% with intra- and inter-day precision lower than 9.94%. This method has already been applied to series of samples from clinical trials, and there was no significant difference between serum and ethylenediaminetetraacetic acid plasma samples. This article is protected by copyright. All rights reserved.
    Keywords:  liquid chromatography-tandem mass spectrometry; plasma; serum; steroid hormones
  3. Food Chem. 2021 Mar 22. pii: S0308-8146(21)00649-X. [Epub ahead of print]356 129643
      A rapid screening method of 70 colorants for regulatory control in dyeable foods was established using ultra-high-performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q/Orbitrap MS) with customized accurate-mass database and mass spectral library. A rapid, high-throughput, and simple sample pretreatment condition with low reagent consumption and high recovery was developed on the basis of ultrasound-assisted extraction and dispersion solid-phase extraction. Rapid screening was conducted by comparing the experimentally measured exact mass of the parent and fragment ions, the isotope pattern, and the retention time with the accurate-mass database and by matching the acquired MS/MS spectra against the mass spectral library. The performance of the method was evaluated in terms of linearity, limits of detection, limits of quantitation, recovery, repeatability, reproducibility, and matrix effect. The proposed method was applied for simultaneous analysis of 70 colorants in seven kinds of dyeable foods, and it exhibited great potential for broad, sensitive, and reliable.
    Keywords:  Accurate-mass database; Colorants; Dyeable foods; Hybrid quadrupole–Orbitrap mass spectrometry; Mass spectral library; Ultra-high-performance liquid chromatography
  4. ACS Omega. 2021 Mar 30. 6(12): 8094-8103
      Recent evidence indicates that tryptophan metabolites and neurotransmitters are potential mediators of the microbiome-gut-brain interaction. Here, a high-resolution ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) assay was developed and validated for quantifying 50 neurotransmitters, tryptophan metabolites, and bacterial indole derivatives in mouse serum, feces, and brain. The lower limit of quantitation for the 50 compounds ranged from 0.5 to 100 nmol/L, and sample preparation procedures were adapted for individual compounds to allow quantitation within linearity of the assay with a correlation coefficient >0.99. Reproducibility was tested by intra- and interday precision and accuracy of analysis: intra- and interday precision at the lower limit of quantitation was less than 20% for all compounds, with over two-thirds of the compounds achieving an interday precision below 10%, while the interday accuracy at the lower limit of quantitation ranged from 82.3 to 128.0% for all compounds. The analyte recovery was assessed based on sample-spiked stable-isotope-labeling standards, illustrating a need to consider matrix-specific recovery discrepancies when performing interorgan comparison. Carryover was evaluated by intermittent solvent blank injection. The assay was successfully applied to determining the concentration profiles of neurotransmitter and tryptophan metabolites in serum, feces, and brain of conventionally raised specific pathogen-free (SPF) C57BL/6 mice. Our method may serve as a useful analytical resource for investigating the roles of tryptophan metabolism and neurotransmitter signaling in host-microbiota interaction.
  5. Rapid Commun Mass Spectrom. 2021 Apr 09. e9096
      RATIONALE: Low speed and low flexibility of most LC-MS/MS approaches in early drug discovery delay sample analysis from routine in vivo studies within the same day. A high-throughput platform for the rapid quantification of drug compounds in various in vivo assays was developed and established in routine bioanalysis.METHODS: Automated selection of an efficient and adequate LC method was realized by autonomous sample qualification for ultrafast batch gradients (9 s/sample) or for fast linear gradients (45 s/sample) if samples required chromatography. The hardware and software components of our Rapid and Integrated Analysis System (RIAS) were streamlined for increased analytical throughput via state-of-the-art automation while maintaining high analytical quality.
    RESULTS: Online decision-making was based on a quick assay suitability test (AST), based on a small and dedicated sample set evaluated by two different strategies. 84% of the acquired data points were within ±30% accuracy and 93% of the deviations between the lower limit of quantitation (LLOQ) values were ≤2-fold compared to standard LC-MS/MS systems. Speed, flexibility and overall automation significantly improved.
    CONCLUSIONS: The developed platform provided an analysis time of only 10 min (batch-mode) and 47 min (gradient-mode) per standard pharmacokinetic (PK) study (62 injections). Automation, data evaluation and results handling were optimized to pave the way for machine learning based on decision-making regarding the evaluation strategy of the AST.
  6. J Chromatogr Sci. 2021 Apr 05. pii: bmab030. [Epub ahead of print]
      New analytical quality by design-oriented HPLC method with multiple response optimization (Derringer's desirability function) was demonstrated for simultaneous analysis of three antidiabetic drugs (metformin hydrochloride/empagliflozin/linagliptin) in a fixed-dose combination. Central composite design was employed for systematic optimization of critical method parameters, namely, % organic phase (X1), aqueous phase pH (X2) and flow rate (X3) while resolution, capacity factor and theoretical plate number as critical analytical attributes. Effective chromatographic separation of title analytes was accomplished on Std. Discovery C18 column at 30°C with mobile phase comprising acetonitrile: phosphate buffer pH 5 (38:62% v/v), pumped at a flow rate of 1 mL/min by isocratic elution pattern and UV detection at 222 nm. The model is rectilinear in the range of 1.0-200, 0.2-40 and 0.1-20 μg/mL at retention times of 3.04, 3.93 and 5.99 min for metformin, empagliflozin and linagliptin, respectively. The method obeyed all validation parameters of ICH Q2(R1) guidelines. The proposed HPLC method was highly robust for method transfer, regulatory flexibility within design space and can be used for assay of pharmaceutical dosage forms comprising these analytes. The proposed method was applied for stability studies of drugs under various stress conditions.
    Keywords:  AQbD; CCD; HPLC; Taguchi-design; antidiabetic drugs
  7. Rapid Commun Mass Spectrom. 2021 Apr 08. e9098
      RATIONALE: Mixed-mode reversed-phase/anion exchange liquid chromatography is useful for separations of mixtures containing anions (e.g. ionized acids). However, when using this form of liquid chromatography with mass spectrometry detection, the bleed of amine-containing hydrolysis products from the columns may cause ion suppression or enhancement.METHODS: Using electrospray ionization tandem quadrupole mass spectrometry detection, we determined the ion suppression or enhancement caused by column bleed for three mixed-mode reversed-phase/weak anion-exchange columns containing stationary phases that differ in chemical structure. Two of the stationary phases are based on silica particles, while the third uses ethylene-bridged hybrid organic/inorganic particles, which have improved hydrolytic stability. Mixtures of acidic and basic analytes were combined with the LC flow postcolumn, both with and without a column, and their mass spectrometry ion signal responses (peak areas) were determined. The ratio of signal response with and without a column is the matrix factor. Positive ion electrospray measurements were carried out using 0.1% formic acid (pH~2.7) as a mobile phase additive, and 10 mM ammonium formate (pH~6.4) was used for negative ion electrospray detection.
    RESULTS: The matrix factors under both positive and negative ionization modes were closest to 1 (0.74 - 1.16) for the hybrid particle-based columns, showing minimal ion suppression or enhancement. In contrast, the silica-based columns gave matrix factors ranging from 0.04 - 1.86, indicating high levels of ion suppression or enhancement. These results may be explained by the differences in the structures of the stationary phases, which affect the relative amounts of hydrolysis products that elute from the columns.
    CONCLUSIONS: The low levels of MS ion suppression or enhancement caused by column bleed from the hybrid particle-based columns should allow for accurate quantitative mass spectrometric detection combined with mixed-mode reversed-phase/weak anion exchange chromatography.
  8. Bioinformatics. 2020 Dec 16. pii: btaa1031. [Epub ahead of print]
      MOTIVATION: Implementing and combining methods from a diverse range of R/Bioconductor packages into 'omics' data analysis workflows represents a significant challenge in terms of standardisation, readability and reproducibility. Here we present an R/Bioconductor package, named struct (Statistics in R using Class-based Templates), which defines a suite of class-based templates that allows users to develop and implement highly standardised and readable statistical analysis workflows. Struct integrates with the STATistics Ontology (STATO) in order to ensure consistent reporting and maximises semantic interoperability. We also present a toolbox, named structToolbox, which includes an extensive set of commonly used data analysis methods that have been implemented using struct. This toolbox can be used to build data-analysis workflows for metabolomics and other omics technologies.AVAILABILITY AND IMPLEMENTATION: struct and structToolbox are implemented in R, and are freely available from Bioconductor ( and, including documentation and vignettes. Source code is available and maintained at
  9. J Sep Sci. 2021 Apr 06.
      Enantiomeric drugs are widely used and play important roles in pharmaceuticals. Ion mobility spectrometry coupled with MS technology provides a unique method for distinguishing the enantiomeric drugs, enantiomeric identification and quantitation in the gas phase. In this study, enantiomeric molecules of ibuprofen and flurbiprofen were clearly recognized by forming host-guest complex ions using trapped ion mobility time-of-flight MS. Ternary complex ions can be produced easily by electrospray ionization of the mixed solutions of ibuprofen, cyclodextrins, and CaCl2 , LiCl, or NaCl, as well as flurbiprofen, cyclodextrins, and CaCl2 . The relative contents of different chiral ibuprofens in a mixed solution were also quantitatively measured. This new method is a simple, effective, and a convenient enantioselective analysis method. This article is protected by copyright. All rights reserved.
    Keywords:  Alkali metal ions; Cyclodextrins; Enantioselective analysis; Ion mobility mass spectrometry; Pharmaceutical molecules
  10. Food Chem. 2021 Mar 23. pii: S0308-8146(21)00653-1. [Epub ahead of print]356 129647
      A simple method for the quantification of 40 mycotoxins in milk was developed. This method is based on a QuEChERS extraction followed by the ultra-high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection, and allows the simultaneous analysis of regulated, emerging, and modified mycotoxins. A sample treatment procedure was optimized to include a concentration step for the analysis of some compounds such as aflatoxin M1. The method was in-house validated in terms of limits of detection (LODs), limits of quantification (LOQs), linearity, recoveries, and precision. LOQs lower than 10 ng/mL were obtained, and recoveries ranged from 61% to 120% with a precision, expressed as the relative standard deviation, lower than 15%. Therefore, acceptable performance characteristics were obtained fulfilling European regulations. The method was successfully applied for the quantification of mycotoxins in raw milk. It can be highlighted high occurrence of beauvericin and enniatins were found in low amounts.
    Keywords:  Milk; Mycotoxin; QuEChERS; UHPLC-MS/MS
  11. J Am Soc Mass Spectrom. 2021 Apr 07.
      Bile acids (BAs) are biomolecules synthesized in the liver from cholesterol and are constituents of bile. The in-vivo BA pool includes more than 50 known diverse BAs which are unconjugated, amino acid conjugated, sulfated, and glucuronidated metabolites. Hemostasis of bile acids is known to be highly regulated and an interplay between liver metabolism, gut microbiome function, intestinal absorption, and enterohepatic recirculation. Interruption of BA homeostasis has been attributed to several metabolic diseases and drug induced liver injury (DILI), and their use as potential biomarkers is increasingly becoming important. Speciated quantitative and comprehensive profiling of BAs in various biomatrices from humans and preclinical animal species are important to understand their significance and biological function. Consequently, a versatile one single bioanalytical method for BAs is required to accommodate quantitation in a broad range of biomatrices from human and preclinical animal species. Here we report a versatile, comprehensive, and high throughput liquid chromatography-high resolution mass spectrometry (LC-HRMS) targeted metabolomics method for quantitative analysis of 50 different BAs in multiple matrices including human serum, plasma, and urine and plasma and urine of preclinical animal species (rat, rabbit, dog, and monkey). The method has been sufficiently qualified for accuracy, precision, robustness, and ruggedness and addresses the issue of nonspecific binding of bile acids to plastic for urine samples. Application of this method includes comparison for BA analysis between matched plasma and serum samples, human and animal species differences in BA pools, data analysis, and visualization of complex BA data using BA indices or ratios to understand BA biology, metabolism, and transport.
  12. J Am Soc Mass Spectrom. 2021 Apr 08.
      Volatile breath metabolites serve as potential disease biomarkers. Online mass spectrometry (MS) presents real-time quantification of breath volatile organic compounds (VOCs). The study aims to assess the relationship between two online analytical mass spectrometry techniques in the quantification of target breath metabolites: selected ion flow tube mass spectrometry (SIFT-MS) and proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS). The two following techniques were employed: (i) direct injection with bag sampling using SIFT-MS and PTR-ToF-MS and (ii) direct injection and thermal desorption (TD) tube comparison using PTR-ToF-MS. The concentration of abundant breath metabolites, acetone and isoprene, demonstrated a strong positive linear correlation between both mass spectrometry techniques (r = 0.97, r = 0.89, respectively; p < 0.001) and between direct injection and TD tube (r = 0.97, r = 0.92, respectively; p < 0.001) breath sampling techniques. This was reflected for the majority of short chain fatty acids and alcohols tested (r > 0.80, p < 0.001). Analyte concentrations were notably higher with the direct injection of a sampling bag compared to the TD method. All metabolites produced a high degree of agreement in the detection range of VOCs between SIFT-MS and PTR-ToF-MS, with the majority of compounds falling within 95% of the limits of agreement with Bland-Altman analysis. The cross platform analysis of exhaled breath demonstrates strong positive correlation coefficients, linear regression, and agreement in target metabolite detection rates between both breath sampling techniques. The study demonstrates the transferability of using data outputs between SIFT-MS and PTR-ToF-MS. It supports the implementation of a TD platform in multi-site studies for breath biomarker research in order to facilitate sample transport between clinics and the laboratory.
    Keywords:  SIFT-MS; breath analysis; direct injection; thermal desorption: PTR-ToF-MS; volatile organic compound