bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2021‒12‒12
thirteen papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Nov 27. pii: S1570-0232(21)00550-X. [Epub ahead of print]1188 123069
      Metabolomics deals with the large-scale analysis of metabolites, belonging to numerous compound classes and showing an extremely high chemical diversity and complexity. Lipidomics, being a subcategory of metabolomics, analyzes the cellular lipid species. Both require state-of-the-art analytical methods capable of accessing the underlying chemical complexity. One of the major techniques used for the analysis of metabolites and lipids is Liquid Chromatography-Mass Spectrometry (LC-MS), offering both different selectivities in LC separation and high sensitivity in MS detection. Chromatography can be divided into different modes, based on the properties of the employed separation system. The most popular ones are Reversed-Phase (RP) separation for non- to mid-polar molecules and Hydrophilic Interaction Liquid Chromatography (HILIC) for polar molecules. So far, no single analysis method exists that can cover the entire range of metabolites or lipids, due to the huge chemical diversity. Consequently, different separation methods have been used for different applications and research questions. In this review, we explore the current use of LC-MS in metabolomics and lipidomics. As a proxy, we examined the use of chromatographic methods in the public repositories EBI MetaboLights and NIH Metabolomics Workbench. We extracted 1484 method descriptions, collected separation metadata and generated an overview on the current use of columns, eluents, etc. Based on this overview, we reviewed current practices and identified potential future trends as well as required improvements that may allow us to increase metabolite coverage, throughput or both simultaneously.
    Keywords:  Lipidomics; Liquid Chromatography-Mass Spectrometry; Metabolomics
  2. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Nov 23. pii: S1570-0232(21)00511-0. [Epub ahead of print]1188 123030
      18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18-OXOF) are important biomarkers for the diagnosis of subtypes of primary aldosteronism. The detection of these three analytes by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is free from structurally similar compounds. The aim of this study was to develop and validate a new LC-MS/MS assay for the simultaneous quantification of 18-OHB, 18-OHF and 18-OXOF in plasma and to establish a reference intervals for apparently healthy population. Plasma samples were prepared by solid phase extraction and separated in an ultra-high performance reversed phase column. MS detection was achieved using a triple quadrupole mass spectrometer in both positive and negative ionization modes. The developed assay was then validated against standard guidelines. We collected 691 plasma samples from apparently healthy individuals (M:398, F:293) to establish the reference intervals. The analytes were separated and quantified within 5 min. The newly developed method demonstrated linearity for the detected steroid concentration in range of 5 to 3000 pg/ml for 18-OXOF (r2 = 0.999) and 20 to 3000 pg/ml for 18-OHB (r2 = 0.997) and 18-OHF (r2 = 0.997). The lower limit of quantification (LLOQ) was 2.5 pg/ml, 20 pg/ml and 20 pg/m for 18-OXOF, 18-OHB and 18-OHF respectively. Specificity, precision, accuracy and stability were tested, and met the requirements of the guidelines. 18-OHB was higher in females than in males, but 18-OHF were higher in males than females. The reference intervals of 18-OHB, 18-OHF and 18-OXOF for both genders together were 90.5-1040.6 pg/ml, 224.4-1685.2 pg/ml, 4.0-70.5 pg/ml, respectively. Age was also an important factor influencing the levels of these three hormones. We have developed a sensitive and reliable method for the simultaneous quantification of 18-OHB, 18-OHF, and 18-OXOF. Our work provides a reference interval for the clinical application of these three steroid hormones.
    Keywords:  18-hydroxycorticosterone; 18-hydroxycortisol; 18-oxocortisol; Liquid chromatography-tandem mass spectrometry
  3. J Chromatogr A. 2021 Nov 11. pii: S0021-9673(21)00802-5. [Epub ahead of print]1661 462680
      As metabolites of the gut microbiome, short-chain fatty acids (SCFAs) played an important role in the diagnosis of the metabolic diseases. Because of the high polarity, high volatility and complex matrix of biological samples, the highly sensitive, selective and accurate method to determine SCFAs remains a major challenge. Herein, a new method for simultaneous quantification of eleven SCFAs by derivatization combined with solid phase microextraction (SPME) and gas chromatography tandem mass spectrometry (GC-MS/MS) was developed. Isobutyl chloroformate coupled with isobutanol was used as the reaction reagent to derivatize SCFAs. The method validation data showed a satisfactory linearity with the linear regression coefficients (R) ranging from 0.9964 to 0.9996. The limit of detection (LOD) of all SCFAs ranges from 0.01 ng·mL-1 to 0.72 ng·mL-1 and the limit of quantification (LOQ) ranges from 0.04 ng·mL-1 to 2.41 ng·mL-1. The intra-day and inter-day precision (RSDs) ranged from 0.65% to 8.92% and 1.62% to 15.61%, respectively. The recovery ranged from 88.10% to 108.71%. Finally, the developed method was successfully used to determine SCFAs in mice fecal sample, and ten of the SCFAs were found in feces of mice, including formic acid.
    Keywords:  Derivatization; Feces of mice; Gas chromatography tandem mass spectrometry; Headspace solid phase microextraction
  4. J Proteome Res. 2021 Dec 07.
      The implication of lipid dysregulation in diseases, toxic exposure outcomes, and inflammation has brought great interest to lipidomic studies. However, lipids have proven to be analytically challenging due to their highly isomeric nature and vast concentration ranges in biological matrices. Therefore, multidimensional techniques such as those integrating liquid chromatography, ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (LC-IMS-CID-MS) have been implemented to separate lipid isomers as well as provide structural information and increased identification confidence. These data sets are however extremely large and complex, resulting in challenges for data processing and annotation. Here, we have overcome these challenges by developing sample-specific multidimensional lipid libraries using the freely available software Skyline. Specifically, the human plasma library developed for this work contains over 500 unique lipids and is combined with adapted Skyline functions such as indexed retention time (iRT) for retention time prediction and IMS drift time filtering for enhanced selectivity. For comparison with other studies, this database was used to annotate LC-IMS-CID-MS data from a NIST SRM 1950 extract. The same workflow was then utilized to assess plasma and bronchoalveolar lavage fluid (BALF) samples from patients with varying degrees of smoke inhalation injury to identify lipid-based patient prognostic and diagnostic markers.
    Keywords:  data annotation; ion mobility spectrometry; lipidomics; smoke inhalation; spectral libraries
  5. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Dec 01. pii: S1570-0232(21)00556-0. [Epub ahead of print]1188 123075
      5-fluorouracil (5-FU) and its oral formulation, capecitabine, are widely used in treating a range of malignancies, either alone or in combination with other antineoplastic drugs. Body surface area-based dosing is used for these agents, despite this approach leading to substantial variability in drug exposure and often resulting in either toxicity or treatment failure. Tailoring therapeutic regimens for individual patients using therapeutic drug monitoring (TDM) has been shown to significantly reduce toxicity and improve cancer outcomes. However, for optimum TDM, sample timing is crucial, along with the need for a venepuncture blood sample to obtain the plasma currently used for 5-FU measurement. In addition to complex blood sample handling requirements, large sample volume and frequent sampling required for pharmacokinetic analysis is another barrier to successfully implementing TDM in a healthcare setting. Microsampling is an alternative collection method to venepuncture, which, combined with the now readily available liquid chromatography mass spectrometry (LC-MS/MS) technology, overcomes the plasma-associated issues. It also has the significant advantage of enabling at home and remote sampling, thus facilitating 5-FU TDM in clinical practice. A LC-MS/MS method for simultaneous measurement of capecitabine, 5'-deoxy-5-fluorocytidine, 5'-deoxy-5-fluorouridine and 5-FU using Mitra® microsampling devices for sample collection was developed. A Shimadzu 8060 LC-MS/MS equipped with electrospray ionisation source interface, operated in positive and negative ion modes, with reversed-phase chromatographic separation was employed for sample analysis. Samples were extracted from Mitra® devices using acetonitrile containing stable isotope-labelled internal standards, sonicated, evaporated under vacuum and resuspended in 0.1 % formic acid before injection into the LC-MS/MS. Chromatographic separation was on a Luna Omega Polar C18 (100 × 2.1 mm, 1.6 µm) column with gradient elution of 0.1 % formic acid in water and acetonitrile. Total run time was 5 min, with the injection volume of 1 µL. The intra and inter-day imprecision ranged from 3.0 to 8.1 and 6.3-13.3 % respectively. Accuracy ranged from 95 -114 % for all analytes. Lower limit of quantification with imprecision of < 19 % and accuracy between 89 and 114 % was 0.05 mg/L for 5-FU and 10 µg/L for other analytes. Assays were linear from 0.05 to 50 mg/L for 5-FU and 10-10,000 µg/L for all other analytes. Analytes were stable on Mitra® devices for up to 9 months at room temperature, 2 years at -30 ℃ and 3 days at 50 ℃. The method was successfully applied for the analysis of samples from patients undergoing cancer treatment with 5-FU and capecitabine. Microsampling using volumetric absorptive microsampling proved to be as reliable as conventional blood collection for 5-FU and capecitabine. This sampling technique may lead to less invasive and better-timed sample collection for TDM, supporting dose optimization strategy.
    Keywords:  5-Fluorouracil; Capecitabine; Liquid chromatography-tandem mass spectrometry; Volumetric absorptive microsampling (VAMS (TM))
  6. Clin Chim Acta. 2021 Dec 03. pii: S0009-8981(21)00422-8. [Epub ahead of print]
      BACKGROUND: Choline, a gut microbiome metabolite, is associated with cardiovascular risk and other chronic illnesses. The aim was to develop a high-throughput nuclear magnetic resonance (NMR)-based assay to measure choline on the Vantera® Clinical Analyzer.METHODS: A non-negative deconvolution algorithm was developed to quantify choline. Assay performance was evaluated using CLSI guidelines.
    RESULTS: Deming regression analysis comparing choline concentrations by NMR and mass spectrometry (n=28) exhibited a correlation coefficient of 0.998 (intercept=-9.216, slope=1.057). The LOQ were determined to be 7.1 µmol/l in serum and 5.9 µmol/l in plasma. The coefficients of variation (%CV) for intra- and inter-assay precision ranged from 6.2-14.8% (serum) and 5.4-11.3% (plasma). Choline concentrations were lower in EDTA plasma by as much as 38% compared to serum, however, choline was less stable in serum compared to plasma. In a population of apparently healthy adults, the reference interval was <7.1-20.0 µmol/l (serum) and <5.9-13.1 µmol/l (plasma). Linearity was demonstrated well beyond these intervals. No interference was observed for a number of substances tested.
    CONCLUSIONS: The newly developed, high-throughput NMR-based assay exhibited good performance characteristics enabling quantification of choline in serum and plasma for clinical use.
    Keywords:  Choline; Nuclear magnetic resonance spectroscopy
  7. Anal Chem. 2021 Dec 07.
      Online, droplet-based in-source chemical derivatization is accomplished using a coaxial-flow contained-electrospray ionization (contained-ESI) source to enhance sensitivity for the mass spectrometric analysis of saccharides. Derivatization is completed in microseconds by exploiting the reaction rate acceleration afforded by electrospray microdroplets. Significant improvements in method sensitivity are realized with minimal sample preparation and few resources when compared to traditional benchtop derivatizations. For this work, the formation of easily ionizable phenylboronate ester derivatives of several mono-, di-, and oligosaccharides is achieved. Various reaction parameters including concentration and pH were evaluated, and a Design of Experiments approach was used to optimize ion source parameters. Signal enhancements of greater than two orders of magnitude were observed for many mono- and disaccharides using in-source phenylboronic acid derivatization, resulting in parts-per-trillion (picomolar) limits of detection. In addition, amino sugars such as glucosamine, which do not ionize in negative mode, were detected at low parts-per-billion concentrations, and isobaric sugars such as lactose and sucrose were easily distinguished. The new in-source derivatization approach can be employed to expand the utility of ESI-MS analysis for compounds that historically experience limited sensitivity and detectability, while avoiding resource-intensive, bulk-phase derivatization procedures.
  8. J Lipid Res. 2021 Dec 01. pii: S0022-2275(21)00141-3. [Epub ahead of print] 100158
      Sex hormones, including androgens, estrogens and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction (LLE) or solid phase extraction (SPE) for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation (CIPS) for sex hormone extraction. After protein-precipitation with acetonitrile (ACN) and adjusting the solution constitution with water, samples were stored at -30° C for 10 minutes to generate two distinct phases: an ACN-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable CIPS-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared to the mainstream LLE-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 minutes. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.
    Keywords:  LC-MS/MS; acetonitrile; cold-induced phase separation; dansyl chloride; derivatization; estradiol; liquid-liquid extraction; sex hormone; solid phase extraction; steroid
  9. Molecules. 2021 Nov 26. pii: 7185. [Epub ahead of print]26(23):
      Mass spectrometry (MS) is an analytical technique that can be used for various applications in a number of scientific areas including environmental, security, forensic science, space exploration, agri-food, and numerous others. MS is also continuing to offer new insights into the proteomic and metabolomic fields. MS techniques are frequently used for the analysis of volatile compounds (VCs). The detection of VCs from human samples has the potential to aid in the diagnosis of diseases, in monitoring drug metabolites, and in providing insight into metabolic processes. The broad usage of MS has resulted in numerous variations of the technique being developed over the years, which can be divided into hyphenated and real-time MS techniques. Hyphenated chromatographic techniques coupled with MS offer unparalleled qualitative analysis and high accuracy and sensitivity, even when analysing complex matrices (breath, urine, stool, etc.). However, these benefits are traded for a significantly longer analysis time and a greater need for sample preparation and method development. On the other hand, real-time MS techniques offer highly sensitive quantitative data. Additionally, real-time techniques can provide results in a matter of minutes or even seconds, without altering the sample in any way. However, real-time MS can only offer tentative qualitative data and suffers from molecular weight overlap in complex matrices. This review compares hyphenated and real-time MS methods and provides examples of applications for each technique for the detection of VCs from humans.
    Keywords:  GC-MS; PTR-MS; SESI-MS; SIFT-MS; chromatography; mass spectrometry
  10. Molecules. 2021 Nov 30. pii: 7288. [Epub ahead of print]26(23):
      Closantel is an antiparasitic drug marketed in a racemic form with one chiral center. It is meaningful to develop a method for separating and analyzing the closantel enantiomers. In this work, two enantiomeric separation methods of closantel were explored by normal-phase high-performance liquid chromatography. The influences of the chiral stationary phase (CSP) structure, the mobile phase composition, the nature and proportion of different mobile phase modifiers (alcohols and acids), and the column temperature on the enantiomeric separation of closantel were investigated in detail. The two enantiomers were successfully separated on the novel CSP of isopropyl derivatives of cyclofructan 6 and n-hexane-isopropanol-trifluoroacetic acid (97:3:0.1, v/v/v) as a mobile phase with a resolution (Rs) of about 2.48. The enantiomers were also well separated on the CSP of tris-carbamates of amylose with a higher Rs (about 3.79) when a mixture of n-hexane-isopropanol-trifluoroacetic acid (55:45:0.1, v/v/v) was used as mobile phase. Thus, the proposed separation methods can facilitate molecular pharmacological and biological research on closantel and its enantiomers.
    Keywords:  chiral stationary phase; closantel; enantiomeric separation; high-performance liquid chromatography; modifiers
  11. J Mass Spectrom. 2021 Dec;56(12): e4798
      Imaging mass spectrometry (IMS) allows the location and abundance of lipids to be mapped across tissue sections of human retina. For reproducible and accurate information, sample preparation methods need to be optimized. Paraformaldehyde fixation of a delicate multilayer structure like human retina facilitates the preservation of tissue morphology by forming methylene bridge crosslinks between formaldehyde and amine/thiols in biomolecules; however, retina sections analyzed by IMS are typically fresh-frozen. To determine if clinically significant inferences could be reliably based on fixed tissue, we evaluated the effect of fixation on analyte detection, spatial localization, and introduction of artifactual signals. Hence, we assessed the molecular identity of lipids generated by matrix-assisted laser desorption ionization (MALDI-IMS) and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) for fixed and fresh-frozen retina tissues in positive and negative ion modes. Based on MALDI-IMS analysis, more lipid signals were observed in fixed compared with fresh-frozen retina. More potassium adducts were observed in fresh-frozen tissues than fixed as the fixation process caused displacement of potassium adducts to protonated and sodiated species in ion positive ion mode. LC-MS/MS analysis revealed an overall decrease in lipid signals due to fixation that reduced glycerophospholipids and glycerolipids and conserved most sphingolipids and cholesteryl esters. The high quality and reproducible information from untargeted lipidomics analysis of fixed retina informs on all major lipid classes, similar to fresh-frozen retina, and serves as a steppingstone towards understanding of lipid alterations in retinal diseases.
    Keywords:  AMD; LC-MS/MS; MALDI-IMS; age-related macular degeneration; fixed; fresh-frozen; lipids; retina
  12. J Chromatogr A. 2021 Nov 22. pii: S0021-9673(21)00804-9. [Epub ahead of print]1661 462682
      Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.
    Keywords:  2D-LC; 3D-LC; APPI-MS; ESI-MS; Lipidomics; Two-dimensional LC
  13. Forensic Sci Res. 2021 ;6(3): 273-280
      A novel hair sample pre-treatment method based on high-speed grinding and solid-phase microextraction (SPME) had been applied for the determination of amphetamines, ketamine and their metabolites in hair samples by liquid chromatography-mass spectrometry (LC-MS). A 20 mg sample of hair was ground with 2 mL of saturated sodium carbonate solution using a high-efficiency hair grinder with 70 Hz oscillation for 2 min at 4 °C. After centrifuging, 1.5 mL of the supernatant was transferred and treated with SPME by direct immersion (DI-SPME). The target analytes extracted by fibre were desorbed and analysed using LC-MS. Under the optimum conditions, a recovery of 90.2%-95.8% was obtained for all analytes. The analytical method was linear for all analytes in the range from 0.2 to 10 ng/mg with the correlation coefficient ranging from 0.9985 to 0.9993. The detection limits for all analytes were estimated to be 0.067 ng/mg. The accuracy (mean relative error) was within ±6.9% and the precision (relative standard error) was less than 6.8%. The combination of high-speed grinding of hair and SPME had the advantages of being easy to perform, environment-friendly and high in detection sensitivity. The proposed method offered an alternative analytical approach for the sensitive detection of drugs in hair samples for forensic purposes.Key PointsThe SPME was involved for the determination of drugs in hair with LC-MS.The hair high-speed grinding combined with SPME was firstly developed.Good linearity, sensitivity, recovery and precision were achieved.
    Keywords:  Forensic sciences; drugs of abuse; forensic toxicology; hair analysis; high-speed grinding; liquid chromatography-mass spectrometry; solid-phase microextraction