bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒08‒08
nineteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Angew Chem Int Ed Engl. 2021 Aug 02.
      The microbiome has a fundamental impact on the human host's physiology through the production of highly reactive compounds that can lead to disease development. One class of such compounds are carbonyl-containing metabolites, which are involved in diverse biochemical processes. Mass spectrometry is the method of choice for analysis of metabolites but carbonyls are analytically challenging. Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at femto- and attomole quantities, and detection of more than 200 metabolites in human fecal, urine and plasma samples. This comprehensive mass spectrometric analysis enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our Chemical Biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.
    Keywords:  Bioorganic Chemistry; Mass Spectrometry; chemoselectivity; metabolites; microbiome
  2. J Adv Pharm Technol Res. 2021 Jul-Sep;12(3):12(3): 267-273
      A wide-range, specific, and precise liquid chromatography tandem mass spectrometric (LC-MS/MS)technique for quantifying fluoxetine (FLX) in human plasma was developed using the RapidTrace® automated solid-phase extraction (SPE) method; the analyte and internal standard (IS) were extricated on Oasis MCX SPE cartridges. Acetonitrile and 5 mM ammonium formate buffer (90:10 v/v) were used as mobile phase to achieve chromatographic separation on the reverse phase (C18 column). The analyte and IS were ionized using +ve electrospray ionization approach which was further traced by multiple-reaction monitoring on a tandem mass spectrometer. To quantify the FLX and FLX-d5, the parent-to-daughter ion transition of m/z of 310.0/44.1 and 315.0/44.0 was used, respectively. The method demonstrated a linear active limit of 0.20-30 ng/ml with recoveries ranging from 63.04% to 79.39% for quality control samples and 61.25% for IS samples. The concentrations over the calibration range demonstrated acceptable precision and accuracy. Due to the high inconsistency of the FLX concentration data, the minimum threshold of the assay was kept at 0.20 ng/ml. The flow rate was maintained at 500 μL/min, and the time for sample analysis for each injection was 3.5 min. The method was found to be specific, sensitive, and faster with minimum utilization of organic solvents and was utilized further for metabolic and pharmacokinetic studies.
    Keywords:  Automation; fluoxetine; human plasma; mass spectrometry; solid-phase extraction
  3. Molecules. 2021 Jul 25. pii: 4487. [Epub ahead of print]26(15):
      Water-soluble B vitamins participate in numerous crucial metabolic reactions and are critical for maintaining our health. Vitamin B deficiencies cause many different types of diseases, such as dementia, anaemia, cardiovascular disease, neural tube defects, Crohn's disease, celiac disease, and HIV. Vitamin B3 deficiency is linked to pellagra and cancer, while niacin (or nicotinic acid) lowers low-density lipoprotein (LDL) and triglycerides in the blood and increases high-density lipoprotein (HDL). A highly sensitive and robust liquid chromatography-tandem mass spectroscopy (LC/MS-MS) method was developed to detect and quantify a vitamin B3 vitamer (nicotinamide) and vitamin B6 vitamers (pyridoxial 5'-phosphate (PLP), pyridoxal hydrochloride (PL), pyridoxamine dihydrochloride (PM), pridoxamine-5'-phosphate (PMP), and pyridoxine hydrochloride (PN)) in human hair samples of the UAE population. Forty students' volunteers took part in the study and donated their hair samples. The analytes were extracted and then separated using a reversed-phase Poroshell EC-C18 column, eluted using two mobile phases, and quantified using LC/MS-MS system. The method was validated in human hair using parameters such as linearity, intra- and inter-day accuracy, and precision and recovery. The method was then used to detect vitamin B3 and B6 vitamers in the human hair samples. Of all the vitamin B3 and B6 vitamers tested, only nicotinamide was detected and quantified in human hair. Of the 40 samples analysed, 12 were in the range 100-200 pg/mg, 15 in the range 200-500 pg/mg, 9 in the range of 500-4000 pg/mg. The LC/MS-MS method is effective, sensitive, and robust for the detection of vitamin B3 and its vitamer nicotinamide in human hair samples. This developed hair test can be used in clinical examination to complement blood and urine tests for the long-term deficiency, detection, and quantification of nicotinamide.
    Keywords:  LC-MS/MS; hair analysis; nicotinamide; vitamers; vitamin; vitamin B3; vitamin B6
  4. Metabolites. 2021 Jul 13. pii: 451. [Epub ahead of print]11(7):
      In the last decade, the field of metabolomics has developed tremendously: it is now possible to routinely measure a wide range of metabolites for many specimens at reduced costs, opening the door to many exciting experiments [...].
  5. Anal Chem. 2021 Aug 06.
      The accurate processing of complex liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) data from biological samples is a major challenge for metabolomics, proteomics, and related approaches. Here, we present the pipelines and systems for threshold-avoiding quantification (PASTAQ) LC-MS/MS preprocessing toolset, which allows highly accurate quantification of data-dependent acquisition LC-MS/MS datasets. PASTAQ performs compound quantification using single-stage (MS1) data and implements novel algorithms for high-performance and accurate quantification, retention time alignment, feature detection, and linking annotations from multiple identification engines. PASTAQ offers straightforward parameterization and automatic generation of quality control plots for data and preprocessing assessment. This design results in smaller variance when analyzing replicates of proteomes mixed with known ratios and allows the detection of peptides over a larger dynamic concentration range compared to widely used proteomics preprocessing tools. The performance of the pipeline is also demonstrated in a biological human serum dataset for the identification of gender-related proteins.
  6. Metab Eng Commun. 2021 Dec;13 e00177
      13C-based metabolic flux analysis (13C-MFA) is an essential tool for estimating intracellular metabolic flux levels in metabolic engineering and biology. In 13C-MFA, a metabolic flux distribution that explains the observed isotope labeling data was computationally estimated using a non-linear optimization method. Herein, we report the development of mfapy, an open-source Python package developed for more flexibility and extensibility for 13C-MFA. mfapy compels users to write a customized Python code by describing each step in the data analysis procedures of the isotope labeling experiments. The flexibility and extensibility provided by mfapy can support trial-and-error performance in the routine estimation of metabolic flux distributions, experimental design by computer simulations of 13C-MFA experiments, and development of new data analysis techniques for stable isotope labeling experiments. mfapy is available to the public from the Github repository (
    Keywords:  13C-based metabolic flux analysis; Experimental design; Non-linear optimization; Open-source software; Python package
  7. Pract Lab Med. 2021 Aug;26 e00246
      Background: Therapeutic drug monitoring (TDM) of gentamicin sulfate (GEN) is usually recommended, particularly in critical patients. Only a few reports had described the determination of GEN in plasma or plasma using LC-MS/MS.Objective: This study aimed to develop and validate a sensitive ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of GEN in small volumes of human plasma.
    Results: The use of a very low concentration of the ion-pairing agent HFBA allowed significant retention of the very polar GEN forms in a reversed phase UHPLC column. The solid-phase extraction (SPE) procedure allowed clean extracts, with no interferences detected in blank samples, and high sensitivity. The assay was linear on the range of 0.2-40 mg L-1 of GEN complex. The combined GEN complex had inter-assay CV of 8.8-10.0%, intra-assay CV of 10.2-11.0%, and accuracy of 96.8-104.0%. The assay was applied to 17 clinical samples obtained from neonate patients. Measured concentrations were in the range of 0.15-3.57 mg L-1 for GEN C1, 0.12-3.55 mg L-1 for GEN C1a, 0.20-5.77 mg L-1 for GEN C2, and 0.47-12.88 mg L-1 for the GEN complex, all within the linear range of the assay.
    Conclusion: A sensitive assay for the quantification of gentamicin in plasma using anion-exchange SPE and UHPLC-MS/MS was validated. The assay can be used for TDM of gentamicin, particularly in centers with access to proper instrumentation and with a low demand for gentamicin measurements, where immunoassays are not cost-effective.
    Keywords:  Gentamicin; Neonates; Solid-phase extraction; Therapeutic drug monitoring; UHPLC-MS/MS
  8. Metabolites. 2021 Jul 13. pii: 452. [Epub ahead of print]11(7):
      NMR spectroscopy is a widely used method for the detection and quantification of metabolites in complex biological fluids. However, the large number of metabolites present in a biological sample such as urine or plasma leads to considerable signal overlap in one-dimensional NMR spectra, which in turn hampers both signal identification and quantification. As a consequence, we have developed an easy to use R-package that allows the fully automated deconvolution of overlapping signals in the underlying Lorentzian line-shapes. We show that precise integral values are computed, which are required to obtain both relative and absolute quantitative information. The algorithm is independent of any knowledge of the corresponding metabolites, which also allows the quantitative description of features of yet unknown identity.
    Keywords:  1D; NMR; deconvolution; metabolites; quantification; signal identification
  9. Int J Mol Sci. 2021 Jul 28. pii: 8054. [Epub ahead of print]22(15):
      We designed a concept of 3D-printed attachment with porous glass filter disks-SLIDE (Sweat sampLIng DevicE) for easy sampling of apocrine sweat. By applying advanced mass spectrometry coupled with the liquid chromatography technique, the complex lipid profiles were measured to evaluate the reproducibility and robustness of this novel approach. Moreover, our in-depth statistical evaluation of the data provided an insight into the potential use of apocrine sweat as a novel and diagnostically relevant biofluid for clinical analyses. Data transformation using probabilistic quotient normalization (PQN) significantly improved the analytical characteristics and overcame the 'sample dilution issue' of the sampling. The lipidomic content of apocrine sweat from healthy subjects was described in terms of identification and quantitation. A total of 240 lipids across 15 classes were identified. The lipid concentrations varied from 10-10 to 10-4 mol/L. The most numerous class of lipids were ceramides (n = 61), while the free fatty acids were the most abundant ones (average concentrations of 10-5 mol/L). The main advantages of apocrine sweat microsampling include: (a) the non-invasiveness of the procedure and (b) the unique feature of apocrine sweat, reflecting metabolome and lipidome of the intracellular space and plasmatic membranes. The SLIDE application as a sampling technique of apocrine sweat brings a promising alternative, including various possibilities in modern clinical practice.
    Keywords:  apocrine sweat; lipidomics; mass spectrometry; microsampling; profiling
  10. Biomed Chromatogr. 2021 Aug 07. e5224
      A liquid chromatography-tandem mass spectrometry method was developed and validated to quantify alectinib, crizotinib, erlotinib and gefitinib. This assay can be combined with our method for osimertinib, allowing quantification of the most used ALK- and EGFR- tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer with a single-assay setup. Chromatographic separation was performed on a HyPURITY® C18 analytical column using an elution gradient of ammonium acetate in water and in methanol, both acidified with formic acid 0,1%. Detection and quantification were performed by a triple quad mass spectrometer with an electrospray ionization interface. This method led to robust results, as the selectivity, carry-over, precision and accuracy met all pre-specified requirements. The assay was validated over a linear range of 100 - 2000 ng/mL for alectinib and erlotinib and 50 - 1000 ng/mL for crizotinib and gefitinib. Alectinib, crizotinib, erlotinib and gefitinib were all stable for at least 4 hours in whole blood (at room temperature (RT) and at 4°C) and for at least one month in EDTA-plasma when stored at -80°C, while osimertinib proved to be unstable at RT. Although high-performance liquid chromatography was used, the run time was short and comparable with other methods using ultra-high performance liquid chromatography.
    Keywords:  LC-MS/MS analysis; non-small cell lung cancer; tyrosine kinase inhibitors; validation
  11. Molecules. 2021 Jul 27. pii: 4514. [Epub ahead of print]26(15):
      Examination of fentanyl levels is frequently performed in certain scientific evaluations and forensic toxicology. It often involves the collection of very variable blood samples, including lipemic plasma or serum. To date, many works have reported the methods for fentanyl detection, but none of them have provided information about the impact on the assay performance caused by an excessive amount of lipids. This aspect may be, however, very important for highly lipophilic drugs like fentanyl. To address this issue, we developed the liquid chromatography method with mass spectrometry detection and utilized it to investigate the impact of lipids presence in rabbit plasma on the analytical method performance and validation. The validation procedure, conducted for normal plasma and lipemic plasma separately, resulted in good selectivity, sensitivity and linearity. The limits of detection and quantification were comparable between the two matrices, being slightly lower in normal plasma (0.005 and 0.015 µg/L) than in lipemic plasma (0.008 and 0.020 µg/L). Liquid-liquid extraction provided a low matrix effect regardless of the lipid levels in the samples (<10%), but pronounced differences were found in the recovery and accuracy. In the normal plasma, this parameter was stable and high (around 100%), but in the lipemic matrix, much more variable and less efficient results were obtained. Nevertheless, this difference had no impact on repeatability and reproducibility. In the present work, we provided reliable, convenient and sensitive method for fentanyl detection in the normal and lipemic rabbit plasma. However, construction of two separate validation curves was necessary to provide adequate results since the liquid-liquid extraction was utilized. Therefore, special attention should be paid during fentanyl quantification that involves lipemic plasma samples purified by this technique.
    Keywords:  fentanyl; high performance liquid chromatography tandem mass spectrometry; lipemia; mass spectrometry
  12. Mol Omics. 2021 Aug 06.
      Metabolomics, especially the large-scale untargeted metabolomics, generates massive amounts of data on a regular basis, which often needs to be filtered, screened, analyzed and annotated via a variety of approaches. Data-dependent-acquisition (DDA) mode including inclusion and exclusion rules for tandem mass spectrometry (MS) is routinely used to perform such analyses. While the parameters of data acquisition are important in these processes, there is a lack of systematic studies on these parameters that can be used in data collection to generate metabolic features for molecular-network (MN) analysis on the Global Natural Products Social Molecular Networking (GNPS) platform. To explore the key parameters that impact the formation and quality of MNs, several data-acquisition parameters for metabolomic studies were proposed in this study. The influences of MS1 resolution, normalized collision energy (NCE), intensity threshold, and exclusion time on GNPS analyses were demonstrated. Moreover, an optimization workflow dedicated to Thermo Scientific QE Hybrid Orbitrap instruments is described, and a comparison of phytochemical contents from two forms of black raspberry extract was performed based on the GNPS MN results. Overall, we expect this study to provide additional thoughts on developing a natural-product-analysis workflow using the GNPS network, and to shed some light on future analyses that utilize similar instrumental setups.
  13. J Chromatogr A. 2021 Jul 24. pii: S0021-9673(21)00504-5. [Epub ahead of print]1653 462380
      Lipidomic samples are complex mixtures of structurally different species of a wide range of concentrations providing challenges in their characterization. In this work, we present a proof of concept for the application of a simple microgradient liquid chromatography device on the detailed analysis of lipid classes. Our lipidomic analysis is based on a lipid class microgradient fractionation of a total lipid extract using an in-house-prepared hydrophilic interaction liquid chromatography microcolumn followed by RP-LC/MS of the collected lipid class fractions. The final fractionation method uses a 40-mm-long microcolumn of 500 μm ID with silica stationary phase obtained from a commercially available chromatographic column and the microgradient of the mobile phase prepared in a microsyringe using methyl tert-butyl ether (MTBE) - methanol - water - ammonium acetate mixtures of various elution strengths. MTBE total lipid extract is directly separated by microgradient elution into lipid classes according to their polarity, which enables the collection of isolated fractions of most lipid classes. The method has been applied to the fractionation of porcine brain extract into nonpolar lipids, hexosylceramides, phosphoethanolamines, phosphocholines, sphingomyelins, and lysophosphocholines classes. Achieved repeatability, recovery, and advanced lipid coverage prove the applicability of the microgradient fractionation of total lipid extract for the comprehensive lipidomic analysis.
    Keywords:  Lipid class fractionation; Lipidomic analysis; Lipidomics; Microgradient separation; Sample preparation
  14. Lab Med. 2021 Aug 05. pii: lmab059. [Epub ahead of print]
      OBJECTIVE: Irinotecan (CPT-11) is an important drug used in the treatment of several solid tumor types. To minimize its toxicity, therapeutic drug monitoring of CPT-11 and its major metabolites (SN-38, SN-38-glucuronide [SN-38G], and APC) has been proposed. We aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of CPT-11 and its major metabolites in plasma.METHODS: Specimen preparation consisted of protein precipitation, evaporation, and reconstitution. Analyses were performed on a C18 column using reverse-phase gradient elution. Electrospray ionization and multiple reaction monitoring in positive mode were used for MS. The following heavy isotope-labeled internal standards were used: CPT-11 D10, SN-38 D3, SN-38G D3, and APC D3.
    RESULTS: We found that CPT-11, SN-38G, and APC eluted at ~4.6 to 4.7 minutes, and SN-38 eluted at ~5.1 to 5.2 minutes. A second peak for SN-38 was detected at ~4.6 to 4.7 minutes. Given that the structure of SN-38 is found in CPT-11, SN-38G, and APC, and in the CPT-11 D10 used here, in-source fragmentation was the likely cause. In addition, we found that a low-level SN-38 impurity was present in CPT-11 D10 and to a lesser extent in SN-38 D3.
    CONCLUSION: When developing methods for CPT-11 and its metabolites, it is important to consider the effects of in-source fragmentation and the choice of internal standards.
    Keywords:  CPT-11; in-source fragmentation; internal standard; liquid chromatography; mass spectrometry; therapeutic drug monitoring
  15. J Fluoresc. 2021 Aug 06.
      In spite of a rapid growth of data processing software, that has allowed for a huge advancement in many fields of chemistry, some research issues still remain problematic. A standard example of a troublesome challenge is the analysis of multi-component mixtures. The classical approach to such a problem consists of separating each component from a sample and performing individual measurements. The advent of computers, however, gave rise to a relatively new domain of data processing - chemometry - focused on decomposing signal recorded for the sample rather than the sample itself. Regrettably, still a very few chemometric methods are practically used in everyday laboratory routines. The Authors believe that a brief 'user-friendly' guide-like article on several 'flagship' algorithms of chemometrics may, at least partly, stimulate an increased interest in the use of these techniques among researchers specializing in many fields of chemistry. In the paper, five different techniques of factor analysis are used for the analysis of a three-component system of fluorophores. These algorithms, applied on the excitation-emission spectra, recorded for the 'unknown' mixture, allowed to unambiguously determine its composition without the need for physical separation of the components. An example of using chemometric methods for physical chemistry research is also provided. For each presented technique of the data analysis, a short description of its theoretical background followed by an example of its practical performance is given. In addition, the Reader is supplemented with a basic information on matrix algebra, detailed experimental 'recipes', reference specialist literature and ready-to-use MATLAB codes.
    Keywords:  Evolving factor analysis; Excitation-emission maps; Fluorescence quenching; Multivariate curve resolution; Rank annihilation factor analysis; Spectral data matrices of mixtures
  16. Curr Opin Biotechnol. 2021 Jul 30. pii: S0958-1669(21)00126-9. [Epub ahead of print]71 115-122
      Single-cell metabolomics (SCM) is currently one of the most powerful tools for performing high-throughput metabolic analysis at the cellular level. The power of single-cell metabolomics to determine the metabolic profiles of individual cells makes it very suitable for decoding cell heterogeneity. SCM bears great potential in cell type identification and differentiation within cell colonies. With the development of various equipment and techniques, SCM analysis has become possible for a wide range of biological samples. Many fields have incorporated this cutting-edge analytic tool to generate fruitful findings. This review article pays close attention to the prevalent techniques utilized in SCM and the exciting new findings and applications developed by studies in phytology, neurology, and oncology using SCM.
  17. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jul 21. pii: S1570-0232(21)00348-2. [Epub ahead of print]1179 122867
      The past decades have seen a rise in the prescription of antipsychotic drugs in the European population, despite the risk of extra-pyramidal, metabolic and cardiac side effects. A multi-analyte liquid chromatography - triple quadrupole mass spectrometry method was developed for the quantification of 38 antipsychotic drugs in plasma. Samples were extracted by a straightforward liquid-liquid extraction with methyl-tertiary-butyl-ether and the compounds of interest were chromatographically separated within 6 min. Calibration curves covered the recommended therapeutic range for all compounds, in addition to sub- and supratherapeutic concentrations for most. The method was successfully validated according to the European Medicines Agency guidelines on bioanalytical method validation. Analysis of medico-legal samples confirmed the relatively common use of the second generation antipsychotics quetiapine and olanzapine, as well as the continued presence of the first generation antipsychotic haloperidol.
    Keywords:  Antipsychotics; Forensic toxicology; Liquid chromatography - tandem mass spectrometry; Therapeutic drug monitoring
  18. Front Physiol. 2021 ;12 699712
      The endocannabinoid system is expressed in cells throughout the body and controls a variety of physiological and pathophysiological functions. We describe robust and reproducible UPLC-MS/MS-based methods for analyzing metabolism of the endocannabinoids, 2-arachidonoyl-sn-glycerol and arachidonoyl ethanolamide, and related monoacylglycerols (MAGs) and fatty acid ethanolamides (FAEs), respectively, in mouse mucosal tissues (i.e., intestine and lung). These methods are optimized for analysis of activity of the MAG biosynthetic enzyme, diacylglycerol lipase (DGL), and MAG degradative enzymes, monoacylglycerol lipase (MGL) and alpha/beta hydrolase domain containing-6 (ABHD6). Moreover, we describe a novel UPLC-MS/MS-based method for analyzing activity of the FAE degradative enzyme, fatty acid amide hydrolase (FAAH), that does not require use of radioactive substrates. In addition, we describe in vivo pharmacological methods to inhibit MAG biosynthesis selectively in the mouse small-intestinal epithelium. These methods will be useful for profiling endocannabinoid metabolism in rodent mucosal tissues in health and disease.
    Keywords:  UPLC-MS/MS; alpha/beta hydrolase domain containing 6; diacylglycerol lipase; endocannabinoids; enzyme kinetics; fatty acid amide hydrolase; lipid metabolism; monoacylglycerol lipase