bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒08‒01
sixteen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. Annu Rev Anal Chem (Palo Alto Calif). 2021 07 27. 14(1): 467-487
      High-resolution mass spectrometry (MS) has advanced the study of metabolism in living systems by allowing many metabolites to be measured in a single experiment. Although improvements in mass detector sensitivity have facilitated the detection of greater numbers of analytes, compound identification strategies, feature reduction software, and data sharing have not kept up with the influx of MS data. Here, we discuss the ongoing challenges with MS-based metabolomics, including de novo metabolite identification from mass spectra, differentiation of metabolites from environmental contamination, chromatographic separation of isomers, and incomplete MS databases. Because of their popularity and sensitive detection of small molecules, this review focuses on the challenges of liquid chromatography-mass spectrometry-based methods. We then highlight important instrumentational, experimental, and computational tools that have been created to address these challenges and how they have enabled the advancement of metabolomics research.
    Keywords:  chromatography; isotope tracing; mass spectrometry; metabolomics; multi-stage mass spectrometry; retention indices
  2. Anal Chem. 2021 Jul 30.
      From microbes to human beings, nontargeted metabolic profiling by liquid chromatography (LC)-mass spectrometry (MS) has been commonly used to investigate metabolic alterations. Still, a major challenge is the annotation of metabolites from thousands of detected features. The aim of our research was to go beyond coverage of metabolite annotation in common nontargeted metabolomics studies by an integrated multistep strategy applying data-dependent acquisition (DDA)-based ultrahigh-performance liquid chromatography (UHPLC)-high-resolution mass spectrometry (HRMS) analysis followed by comprehensive neutral loss matches for characteristic metabolite modifications and database searches in a successive manner. Using pooled human urine as a model sample for method establishment, we found 22% of the detected compounds having modifying structures. Major types of metabolite modifications in urine were glucuronidation (33%), sulfation (20%), and acetylation (6%). Among the 383 annotated metabolites, 100 were confirmed by standard compounds and 50 modified metabolites not present in common databases such as human metabolite database (HMDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were structurally elucidated. Practicability was tested by the investigation of urines from pregnant women diagnosed with gestational diabetes mellitus vs healthy controls. Overall, 83 differential metabolites were annotated and 67% of them were modified metabolites including five previously unreported compounds. To conclude, the systematic modifying group-assisted strategy can be taken as a useful tool to extend the number of annotated metabolites in biological and biomedical nontargeted studies.
  3. Anal Chem. 2021 Jul 28.
      The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.
  4. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Apr 24. pii: S1570-0232(21)00205-1. [Epub ahead of print]1179 122725
      One predominant and bioactive folate vitamer circulating in the blood is 5-methyltetrahydrofolate (5-Me-THF). In this study, a method for the accurate determination of 5-Me-THF in human plasma samples of various volumes was established using isotope dilution ultra-high performance liquid chromatography-mass spectrometry (ID-UPLC-MS). For this purpose, 500 μL of homogeneous human plasma was initially employed, and the 5-Me-THF and the 13C5-5-Me-THF standard solutions prepared using 1% ascorbic acid in water gave the calibration solution and spiking sample. The desired amount of 13C5-5-Me-THF standard solution was spiked into the sample followed by sample pretreatment. The method was validated for its repeatability, reproducibility, recovery, and limits of detection and quantification. Subsequently, it was applied to smaller volumes of human plasma samples (i.e., 50 and 10 μL), the results of which corresponded well with those obtained using 500 μL. The feasibility of the method was further confirmed using 10 μL of a standard reference material, SRM 3949, which is a human serum sample containing three different levels of 5-Me-THF. The established ID-UPLC-MS method was successfully applied to various volumes of human plasma or serum ranging from 500 to 10 μL, which exhibited particularly good sensitivity in addition to reliable results for the quantification of 5-Me-THF. Our method therefore expands on the ability to obtain accurate quantitative results for 5-Me-THF using small volumes of blood.
    Keywords:  5-Methyltetrahydrofolate; Human plasma; Isotope dilution mass spectrometry; Quantification; Ultra-high-performance liquid chromatography
  5. Sci Total Environ. 2021 Jul 17. pii: S0048-9697(21)04084-5. [Epub ahead of print]798 149012
      Liquid separation coupled to mass spectrometry is often used for non-targeted analyses in various fields, such as metabolomics. However, the combination of non-standardized methods, various mass spectrometers (MS) and processing tools for data evaluation affect biomarker discovery potentially. Here, we present a comprehensive study of these factors based on non-targeted liquid chromatography coupled to time-of-flight (TOF) and Orbitrap MS and capillary zone electrophoresis to Orbitrap analyses of the same bioreactor samples, describing the correlation of its gas yield with changing feature signal intensity. The three datasets were processed with MZmine 2 and XCMS online and subsequential Partial Least Square Regression (PLSR) with Variable Importance in Projection (VIP) ranking for feature prioritization. The six feature tables were compared to evaluate their overlap of shared features and the influence of the processing software and MS instrument on the VIP values and fold changes. The overlaps, defined as a fraction of one feature table found in the comparative table, were from 27% to 57% for the comparison of MZmine and XCMS and from 15% to 50% between Orbitrap and TOF data sets, respectively. Considering the most relevant features only (VIP >1.5), the overlaps were increased significantly in all cases from 26% to 95%. For the same data set, both VIP values and fold changes were well correlated, however, varied significantly between TOF and Orbitrap. CE-MS showed higher total feature numbers compared to LC-MS, most likely due to its more appropriate selectivity, different sample preparation, and/or the sensitive nano-ESI interface. Since only less than 10% of MS/MS data overlapped, CE-MS provided complementary information to LC-MS. Overall, our systematic study proves the benefits of using different separation techniques and processing tools but also indicates a significant influence of mass spectrometry on comprehensive biomarker discovery.
    Keywords:  Biogas plant; Bioreactor; Capillary electrophoresis; Liquid chromatography; Mass spectrometry; Non-targeted analysis
  6. Anal Chem. 2021 Jul 28.
      Untargeted metabolomics using liquid chromatography coupled to mass spectrometry (LC-MS) allows the detection of thousands of metabolites in biological samples. However, LC-MS data annotation is still considered a major bottleneck in the metabolomics pipeline since only a small fraction of the metabolites present in the sample can be annotated with the required confidence level. Here, we introduce mWISE (metabolomics wise inference of speck entities), an R package for context-based annotation of LC-MS data. The algorithm consists of three main steps aimed at (i) matching mass-to-charge ratio values to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, (ii) clustering and filtering the potential KEGG candidates, and (iii) building a final prioritized list using diffusion in graphs. The algorithm performance is evaluated with three publicly available studies using both positive and negative ionization modes. We have also compared mWISE to other available annotation algorithms in terms of their performance and computation time. In particular, we explored four different configurations for mWISE, and all four of them outperform xMSannotator (a state-of-the-art annotator) in terms of both performance and computation time. Using a diffusion configuration that combines the biological network obtained from the FELLA R package and raw scores, mWISE shows a sensitivity mean (standard deviation) across data sets of 0.63 (0.07), while xMSannotator achieves a sensitivity of 0.55 (0.19). We have also shown that the chemical structures of the compounds proposed by mWISE are closer to the original compounds than those proposed by xMSannotator. Finally, we explore the diffusion prioritization separately, showing its key role in the annotation process. mWISE is freely available on GitHub ( under a GPL license.
  7. Biomed Chromatogr. 2021 Jul 29. e5220
      To explore the metabolic effect of chemerin, adipose-specific chemerin knockout (adipo-chemerin (-/-)) male mice were established and fed with 5-week normal diet (ND) or high fat diet (HFD), then the glycolipid metabolism index were measured and epididymal adipose tissue metabolomics were detected using untargeted liquid-chromatography-tandem mass spectrometry (LC-MS). Under HFD, adipo-chemerin (-/-) mice showed improved glycolipid metabolism (decreases of TC, LDL, insulin and HOMA-IR) compared with flox (control) mice. Furthermore, OPLS-DA score plots found metabolites separations between adipo-chemerin (-/-) mice and flox mice fed with ND and HFD. Under HFD, 28 metabolites were significantly enhanced in adipo-chemerin (-/-) mice, and pathway enrichment analysis suggested strong relationship of the differential metabolites with arginine and proline metabolism, phenylalanine metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis, which were directly or indirectly related to lipid metabolism, inflammation and oxidative stress. Under ND, taurine was increased in adipo-chemerin (-/-) mice, involving in taurine and hypotaurine metabolism and primary bile acid biosynthesis. In conclusion, the improved effect of chemerin knockdown on the glycolipid metabolism of HFD-feeding male mice might be associated with the increases of differential metabolites and metabolic pathways involving in lipid metabolism, inflammation and oxidative stress, which provided insights into the mechanism of chemerin from metabolomics aspect.
    Keywords:  LC-MS; chemerin; epididymal adipose; high fat diet; metabolomics
  8. J Pharm Biomed Anal. 2021 Jul 24. pii: S0731-7085(21)00393-9. [Epub ahead of print]204 114282
      We developed and validated a new rapid and sensitive gas chromatography-tandem mass spectrometry method for the determination of cocaine and its metabolites benzoylecgonine, norcocaine, ecgonine methyl esther and cocaethylene in hair of consumers. Hair samples were firstly decontaminated with three subsequent dichloromethane washes, then incubated for one hour with M3® buffer to promote analytes solubilization and stabilization and finally solid phase extracted. All extracts were derivatized and injected into GC-MS/MS with electron impact ionization. Multiple Reaction Monitoring was used for the acquisition of characteristic analytes ion transitions reaching a high sensitivity 0.01 ng/mg COC and metabolites limit of quantification. The method was linear in the COC and metabolites calibration ranges (LLOQ-10 ng/mg and LLOQ-1 ng/mg, respectively). Intra-assay and inter-assay precision were always lower than 15 %, accuracy never exceeded ± 6.6 %. The main advantages of the presented method are the fast, simple and innovative pretreatment procedure together with the instrumental sensitivity that allowed to measure also less concentrated metabolites.
    Keywords:  COC; COC metabolites; Drugs hair test; GC–MS/MS; MRM
  9. Ann Clin Biochem. 2021 Jul 29. 45632211038021
      BACKGROUND: Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first line investigation for gastrointestinal neuroendocrine tumours (NETs) that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response.AIM: To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-HIAA that incorporates a supported liquid extraction (SLE) and 13C labelled internal standard.
    METHODS: Samples were diluted in ammonium acetate containing a 13C labelled internal standard (5-Hydroxyindole-3a,4,5,6,7,7a-13C6-3-acetic acid). SLE was performed followed by chromatographic separation using the 2.1 x 30 mm CORTECS® UPLC® T3 column. Mass spectrometry detection (Waters Xevo TQ-XS) was performed in electrospray positive mode using the transitions 192.3> 146.4 m/z (quantifier) and 192.3>118.4 m/z (qualifier) for 5-HIAA and 198.2 > 152.4 m/z for 13C-5-HIAA.
    RESULTS: A well-defined 5-HIAA peak was observed at 0.8 min with a run time of 2.4 mins. The assay was linear (r2 >0.99) to 382 µmol/L, with a lower limit of quantification of 5.3 µmol/L (CV <15%). Analysis of 29 external quality assurance (EQA) samples showed good agreement between our method and the UKNEQAS method mean (4.7% positive bias). The intra and inter-assay precision was within acceptable limits and the assay was stable up to 96 hrs post-extraction with minimal carryover.
    CONCLUSION: We have developed a robust LC-MS/MS method with semi-automated extraction that offers an improved run time and performance over the existing, labour intensive, HPLC method. The method was quick, precise, showed good agreement with UKNEQAS EQA material and is in routine service for clinical samples.
    Keywords:  Chromatography; Mass spectrometry; Tumour markers
  10. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jul 06. pii: S1570-0232(21)00297-X. [Epub ahead of print]1179 122816
      A simple, sensitive, rapid and specific method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the simultaneous quantification of dantrolene (DAN) and paracetamol (PAR) in real human plasma was developed and validated. The preparation of sample was achieved by liquid-liquid extraction with tertiary butyl methyl ether. The analysis was performed on a reversed-phase C18column (1.7 µm, 2.1 × 30 mm) using acetonitrile: 0.1% formic acid (80:20, v/v) as the mobile phase and pumped in an isocratic mode at a flow rate of 0.3 mL/min using citalopram (CIT) as an internal standard. Tandem mass spectrometric detection was carried out by both positive and negative electrospray ionization (ESI) in the multiple-reaction monitoring mode (MRM). The analysis was carried out within 1 min for each sample which made it possible to analyze more than 350 human samples per day. Validation of the method was performed according to FDA guidelines for bio-analytical method. The method was found to be linear in the range of 25-2500 ng/mL and 100-10,000 ng/mL for DAN and PAR, respectively. The method was applied successfully for the determination of the two analytes in the plasma after oral administration of Dantrelax® compound capsules to healthy volunteers. The study was accomplished after approval of the ethics committee.
    Keywords:  Bioanalytical method validation; Dantrelax® compound capsules; Dantrolene; Egyptian healthy volunteers; Paracetamol; UPLC–MS/MS
  11. Leg Med (Tokyo). 2021 Jul 09. pii: S1344-6223(21)00103-6. [Epub ahead of print]53 101939
      A simple and sensitive liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of 3-chloro-L-tyrosine (Cl-Tyr) was developed and validated. For sample preparation, 50 μL of the body fluids or tissue extracts were processed by protein precipitation followed by the derivatization with dansyl chloride. The calibration curve was linear over the concentration range of 2.0-200 ng/mL blood or 4.0-400 ng/g tissue. Our method allowed the reproducible and accurate quantification. That is, the intra- and inter-assay coefficients of variation were below 7.73 and 6.94%, respectively in both the blood and lung. We applied the developed method to the analysis of Cl-Tyr in the human autopsy samples, which were suspected of chlorine poisoning, and detected 55.2 ng/mL and 206.6 ng/g Cl-Tyr in left heart blood and lung, respectively. Furthermore, in more than 20 autopsy samples, which were obtained from other causes of death including burn, drowning, hanging, internal disease, trauma and drug poisoning, Cl-Tyr was almost not detected in their both body fluids and organ tissues. In conclusion, the data here reported demonstrate that the LC/ESI-MS/MS method allows the Cl-Tyr in the autopsy samples and that chlorine exposure strongly affects its level, providing a basis for novel identification tool of chlorine poisoning.
    Keywords:  3-Chloro-L-tyrosine; Chlorine poisoning; Dansyl chloride; Derivatization; LC–MS
  12. Nat Protoc. 2021 Jul 28.
      Metabolic phenotyping is an important tool in translational biomedical research. The advanced analytical technologies commonly used for phenotyping, including mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, generate complex data requiring tailored statistical analysis methods. Detailed protocols have been published for data acquisition by liquid NMR, solid-state NMR, ultra-performance liquid chromatography (LC-)MS and gas chromatography (GC-)MS on biofluids or tissues and their preprocessing. Here we propose an efficient protocol (guidelines and software) for statistical analysis of metabolic data generated by these methods. Code for all steps is provided, and no prior coding skill is necessary. We offer efficient solutions for the different steps required within the complete phenotyping data analytics workflow: scaling, normalization, outlier detection, multivariate analysis to explore and model study-related effects, selection of candidate biomarkers, validation, multiple testing correction and performance evaluation of statistical models. We also provide a statistical power calculation algorithm and safeguards to ensure robust and meaningful experimental designs that deliver reliable results. We exemplify the protocol with a two-group classification study and data from an epidemiological cohort; however, the protocol can be easily modified to cover a wider range of experimental designs or incorporate different modeling approaches. This protocol describes a minimal set of analyses needed to rigorously investigate typical datasets encountered in metabolic phenotyping.
  13. J Pharm Biomed Anal. 2021 Jul 21. pii: S0731-7085(21)00385-X. [Epub ahead of print]204 114274
      JQ1, is a cell-permeable small-molecule inhibitor of bromodomain and extra-terminal protein (BET) function with reportedly good CNS penetration, however, unbound and pharmacologically active CNS JQ1 exposures have not been characterized. Additionally, no quantitative bioanalytical methods for JQ1 have been described in the literature to support the CNS penetration studies. In the present article, we discuss the development and validation of a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative methods to determine JQ1 in mouse plasma and brain microdialysate. JQ1 and the internal standard, dabrafenib (ISTD), were extracted from plasma and microdialysate samples using a simple solid phase extraction protocol performed on an Oasis HLB μElution plate. Chromatographic separation of JQ1 and ISTD was achieved on a reversed phase C12 analytical column with gradient elution profile of mobile phases (MP A: water containing 0.1 % formic acid and MP B: acetonitrile containing 0.1 % formic acid) at a flow rate of 0.6 mL/min. The mass spectrometric detection was performed in the positive MRM ion mode by monitoring the transitions 457.40 > 341.30 (JQ1) and 520.40 > 307.20 (ISTD). The calibration curves demonstrated good linearities over the concentration range of 5-1000 ng/mL for the mouse plasma method (r2 ≥ 0.99) and 0.5-500 ng/mL for the microdialysate method (r2 ≥ 0.99). The experimental limit of quantification obtained was 5 and 0.5 ng/mL for the mouse plasma and microdialysate method, respectively, with the coefficient of variation less than 10 % for the analyte peak area. All the other validation parameters, including intra-and inter-day accuracy and precision, matrix effect, selectivity, carryover effect, and stability, were within the USFDA bioanalytical guidelines acceptance limits. The LC-MS/MS method was successfully applied to a mouse pharmacokinetic and cerebral microdialysis study to characterize the unbound JQ1 exposure in brain extracellular fluid and plasma.
    Keywords:  BET inhibitor; Cerebral microdialysis; JQ1; LC–MS/MS; Pharmacokinetics; Solid phase extraction
  14. Anal Chem. 2021 Jul 27.
      Single quadrupole mass spectrometry (MS) with enhanced in-source multiple fragment ion monitoring was designed to perform high sensitivity quantitative mass analyses. Enhanced in-source fragmentation amplifies fragmentation from traditional soft electrospray ionization producing fragment ions that have been found to be identical to those generated in tandem MS. We have combined enhanced in-source fragmentation data with criteria established by the European Union Commission Directive 2002/657/EC for electron ionization single quadrupole quantitative analysis to perform quantitative analyses. These experiments were performed on multiple types of complex samples that included a mixture of 50 standards, as well as cell and plasma extracts. The dynamic range for these quantitative analyses was comparable to triple quadrupole multiple reaction monitoring (MRM) analyses at up to 5 orders of magnitude with the cell and plasma extracts showing similar matrix effects across both platforms. Amino acid and fatty acid measurements performed from certified NIST 1950 plasma with isotopically labeled standards demonstrated accuracy in the range of 91-110% for the amino acids, 76-129% for the fatty acids, and good precision (coefficient of variation <10%). To enhance specificity, a newly developed correlated ion monitoring algorithm was designed to facilitate these analyses. This algorithm autonomously processes, aligns, filters, and compiles multiple ions within one chromatogram enabling both precursor and in-source fragment ions to be correlated within a single chromatogram, also enabling the detection of coeluting species based on precursor and fragment ion ratios. Single quadrupole instrumentation can provide MRM level quantitative performance by monitoring/correlating precursor and fragment ions facilitating high sensitivity analysis on existing single quadrupole instrumentation that are generally inexpensive, easy to operate, and technically less complex.
  15. Front Mol Biosci. 2021 ;8 682134
      Blood is a rich biological sample routinely collected in clinical and epidemiological studies. With advancements in high throughput -omics technology, such as metabolomics, epidemiology can now delve more deeply and comprehensively into biological mechanisms involved in the etiology of diseases. However, the impact of the blood collection tube matrix of samples collected needs to be carefully considered to obtain meaningful biological interpretations and understand how the metabolite signatures are affected by different tube types. In the present study, we investigated whether the metabolic profile of blood collected as serum differed from samples collected as ACD plasma, citrate plasma, EDTA plasma, fluoride plasma, or heparin plasma. We identified and quantified 50 metabolites present in all samples utilizing nuclear magnetic resonance (NMR) spectroscopy. The heparin plasma tubes performed the closest to serum, with only three metabolites showing significant differences, followed by EDTA which significantly differed for five metabolites, and fluoride tubes which differed in eleven of the fifty metabolites. Most of these metabolite differences were due to higher levels of amino acids in serum compared to heparin plasma, EDTA plasma, and fluoride plasma. In contrast, metabolite measurements from ACD and citrate plasma differed significantly for approximately half of the metabolites assessed. These metabolite differences in ACD and citrate plasma were largely due to significant interfering peaks from the anticoagulants themselves. Blood is one of the most banked samples and thus mining and comparing samples between studies requires understanding how the metabolite signature is affected by the different media and different tube types.
    Keywords:  NMR spectroscopy; anticoagulants; epidemiological studies; metabolic profile; metabolomics; plasma; serum
  16. Bio Protoc. 2021 Jul 05. 11(13): e4081
      The diversity of lipid structures, properties, and combinations in biological tissues makes their extraction and analysis an experimental challenge. Accordingly, even for one of the simplest single-cellular fungi, the budding yeast (Saccharomyces cerevisiae), numerous extraction and analysis protocols have been developed to separate and quantitate the different molecular lipid species. Among them, most are quite sophisticated and tricky to follow. Herein, we describe a yeast total lipids extraction procedure with a relatively good yield, which is appropriate for subsequent thin-layer chromatography (TLC) or liquid chromatography-mass (LC-MS) analysis. We then discuss the most widely used solvent systems to separate yeast phospholipids and neutral lipids by TLC. Finally, we describe an easy and rapid method for silica gel staining by a Coomassie Brilliant Blue-methanol mixture. The stained lipid species can then be quantitated using imaging software such as ImageJ. Overall, the methods described in this protocol are time-saving and novice-friendly.
    Keywords:  Budding yeast; Lipid extraction; Lipid staining; Neutral lipid; Phospholipid; TLC