Metabolites were identified based on an in house library based on authentic analytical standards.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse-phase","Liquid Chromatography MS - positive - reverse-phase"],"chromatography_protocol":["A Bruker Bio-AQ, 50 x 2.1 mm, 2 µm column was used at an oven temperature of 55 °C. At a flow rate of 0.9 mL/min, the mobile phases A (water with 0.1% HCOOH) and B (acetonitrile with 0.1% HCOOH) were delivered with the following gradient: starting conditions 20 % B, 1.5 min 80% B, 1.7 min 100 % B, 1.9 min 100 % B, 1.92 min 20 % B, ending at 2.0 min 20 % B.
A Bruker Bio-AQ, 50 x 2.1 mm, 2 µm column (1901674, Bruker, Bremen, Germany) was used at an oven temperature of 55°C. At a flow rate of 0.9 mL/min, the mobile phases consisted of A (water with 0.1 % HCOOH) and B (acetonitrile with 0.1% HCOOH), and were delivered with the following gradient: starting conditions 20 % B, 0.2 min 20 % B, 1.2 min 80 % B, 1.25 min 100 % B, 1.45 min 100 % B, 1.5 min 20 % B, ending at 1.6 min 20 %.
A Kinetex Biphenyl, 50 x 2.1 mm, (Phenomenex, Torrance, California, United States) 2.6 µm column was used at an oven temperature of 55°C. At a flow rate of 0.9 mL/min, the mobile phases A (water with 0.1% HCOOH) and B (acetonitrile with 0.1% HCOOH) were delivered with the following gradient: starting conditions 0.1 % B, 0.5 min 0.1 % B, 0.9 min 0.15% B, 1.25% 0.15% B, 1.6 min, 40% B, 1.85 min 100 % B, 1.95 min 100 % B, ending at 2.05 min 0.1 % B.
"],"publication":["Rapid chromatography combined with trapped ion mobility mass spectrometry for analysis of derivatized small molecules."],"submitter_name":["Matthias Anagho-Mattanovich"],"submitter_affiliation":["University of Copenhagen"],"organism_part":["Cell Line","blood plasma","reference compound","feces"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["For metabolite extraction, cells were quenched in ice-cold 90% methanol. Culture media were transferred to tubes, wells were rinsed with 1 mL in-house PBS, and 500 µL methanol solution containing 25 ng/µL of 13C3-lactic acid, 13C3-citric acid, 13C4-malic acid (Cambridge Isotope Laboratories, MA, USA), D6-alpha-ketoglutaric acid, D4-succinic acid and D4-fumaric acid (Merck-Sigma Aldrich, MO, USA) as internal standards were added. Cells were scraped, collected, snap-frozen in liquid nitrogen, and subjected to three freeze-thaw-vortex cycles. After 1 h incubation on ice for protein precipitation, samples were centrifuged (16,000 g, 15 min, 4 °C), and supernatants were stored at -80 °C. For subsequent NPH derivatization, 20 µL of supernatant were used.
Short-chain fatty acids were extracted from approximately 100 mg human fecal samples (for experimental- and sample-specific details see the referenced studies) with 1.5 mL extraction mix of 10% methanol containing isotopically labelled internal standards (340 µM acetic acid-D4, propionic acid-D6, and butyric acid-D8, Merck Sigma-Aldrich, MO, USA). The samples were vortexed vigorously for 30 sec before centrifugation at 4,000 rpm at 4°C for 15 min. Samples were thereafter diluted 1:20 with extraction mixture.
Metabolite extraction of TCA and amino acids from plasma was achieved by adding 300 μL of 90% MeOH to 50 µL of NIST® SRM® 1950 reference plasma or Sigma serum H4522 (Merck-Sigma Aldrich, MO, USA), Samples were vortexed and kept on ice for 30 min. Thereafter, the extracts were centrifuged at 18,000 x g at 4 °C for 15 min. Then, 50 µL of the supernatant were transferred to LC-MS vials for amino acid analysis, and 150 µL were transferred to another LC-MS vial for analysis of TCA-metabolites. Prior to drying the LC-MS vials under N2 (stream flow = 6 L/min) for 1 h at room temperature, stable-isotope labeled internal standards were added to the vials. For amino acid analysis, 20 μL of deuterated amino acid standard mixture (SMB00917, Merck-Sigma Aldrich, MO, USA) was added with the following concentrations: alanine-D4 164 µM, arginine-D7 48 µM, asparagine-D3 48 µM, aspartic acid-D3 20 µM, glutamic acid-D5 64 µM, glutamine-D5 222 µM, glycine-D2 152 µM, histidine-D5 50 µM, isoleucine-D10 124 µM, leucine-D10 124 µM, lysine-D8 70 µM, methionine-D8 24 µM, ornithine-D2 51 µM, phenylalanine-D8 44 µM, proline-D7 84 µM, serine-D3 60 µM, threonine-D5 81 µM, tryptophan-D8 31 µM, tyrosine-D7 45 µM, valine-D8 156 µM. For TCA metabolite analysis 20 μL of a mixture of 4000 ng 13C3-lactic acid, 400 ng 13C3-citric acid, 100 ng 13C4-malic acid (Cambridge Isotope Laboratories, MA, USA), 400 ng 13C3-pyruvic acid 100 ng D6-alpha-ketoglutaric acid, 50 ng D4-succinic acid and 50ng D4-fumaric acid (Merck-Sigma Aldrich, MO, USA) were added.
For analysis of SCFAs in plasma, 20 µL of plasma (for experimental- and sample-specific details see the referenced study) were mixed with 20 µL of internal standard mixture in methanol containing isotopically labelled internal standards (100 µM acetic acid-D4, 2 µM propionic acid-D6, and 2 µM butyric acid-D8, Merck Sigma-Aldrich, MO, USA) with a Bravo Automated Liquid Handling Platform (Agilent Technologies, Santa Clara, CA, USA).
"],"organism":["Mus musculus","reference compound","Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS14813"],"author":["Matthias Anagho-Mattanovich. University of Copenhagen. matthias.mattanovich@sund.ku.dk.","Thomas Moritz. University of Copenhagen. thomas.moritz@sund.ku.dk."],"data_transformation_protocol":["Data were processed with Metaboscape 2026 and TASQ 2026 (Bruker, Daltonics GmbH, & Co. KG, Bremen, Germany) for untargeted and targeted investigation, respectively. All processing used optimised software settings for the different analyses. The smoothing parameter for chromatograms in TASQ was always set to 1 Gaussian smoothing cycle, with smoothing width of 1s. Quantification was based on internal calibration curves, ranging for TCA-metabolites from 0.05 to 100 µM, SCFA from 0.1 to 1000 µM, and amino acids 0.1 to 500 µM.
"],"study_factor":["Chromatography method development"],"submitter_email":["matthias.mattanovich@sund.ku.dk"],"sample_collection_protocol":["The brown adipocyte samples were prepared the same way as described in Anagho-Mattanovich et al. 2025. In brief, immortalized murine brown preadipocytes were cultured in 6-well plates using DMEM (Thermo Fisher Scientific) supplemented with 10 % FBS and 1 % penicillin-streptomycin at 37 °C, 5 % CO2. Upon reaching confluence (day 0), differentiation was induced with 20 nM insulin, 1 nM triiodothyronine (T3), 1 µM dexamethasone, 0.125 mM indomethacin, and 0.5 mM isobutylmethylxanthine (Sigma-Aldrich). After 2 days, the medium was replaced with DMEM containing 20 nM insulin and 1 nM T3, refreshed every 2 days. Experiments were performed on day 7 using cells confirmed to be free of mycoplasma. For stimulation, cells were treated with 1 µM L-(-)-norepinephrine (+)-bitartrate salt monohydrate (Sigma-Aldrich), and samples were collected for metabolomics without stimulation (0 h samples) and at 4, 8, and 24 h post-stimulation. For each condition, 6 replicates were collected. An un-inoculated plate was treated the same way as the samples and used as an experimental blank.
Short-chain fatty acids were extracted from approximately 100 mg human fecal samples.
Amino acids were extracted from 50 µL of NIST® SRM® 1950 reference plasma.
"],"omics_type":["Metabolomics"],"study_design":["ultra-performance liquid chromatography-mass spectrometry","Cell Line","Metabolomics","Mus musculus","untargeted analysis","Homo sapiens","derivatization","certified reference material","reference compound mix","Method Development","experimental sample","reference compound","blood plasma","Bruker timsTOF Pro 2","Agilent 1290 Infinity II UHPLC","ion mobility MS","feces"],"curator_keywords":["ultra-performance liquid chromatography-mass spectrometry","Cell Line","Metabolomics","Mus musculus","untargeted analysis","Homo sapiens","derivatization","certified reference material","reference compound mix","Method Development","experimental sample","reference compound","blood plasma","Bruker timsTOF Pro 2","Agilent 1290 Infinity II UHPLC","ion mobility MS","feces"],"mass_spectrometry_protocol":["All mass spectrometry measurements were performed using a Bruker timsTOF Pro 2 (Bruker Daltonics GmbH & Co. KG, Bremen, Germany) instrument equipped with a trapped-ion mobility spectrometry (TIMS) tunnel coupled to a hybrid quadrupole time-of-flight mass spectrometer (TOF-MS). Acquisition was controlled by timsControl 7.05 and HyStar 6.4.
Acquisition was made with a Vacuum Insulated Probe Heated ElectroSpray Ionization source (VIP-HESI) operated in negative (for SCFA and TCA analysis) or positive ionization (for amino acid analysis) mode. Capillary voltage was 3.6 kV (negative mode) or 4 kV (positive mode), nebulizer 4 bar, dry gas flow 8 L/min, dry gas temperature 230 °C, sheath gas temperature 400 °C and sheath gas flow 4 L/min. For the analysis with lower flow-rates (standard methods), the nebulizer was set to 2.5 bar.
Mass range was m/z 50-1200, 1/K0 between 0.45-1.38, and ramp time, 75 ms. The ion charge control was enabled and set to 5.0M. For PASEF, the collision energy was set to 35 eV.
"],"metabolite_name":["fumaric acid","citric acid","succinic acid","2-oxoglutarate","lactate","pyruvic acid"],"additional_accession":[]},"is_claimable":false,"name":"Rapid chromatography combined with trapped ion mobility mass spectrometry for analysis of derivatized small molecules","description":"Liquid chromatography (LC) separates biomolecules based on their physicochemical properties. Reproducible retention times support compound identification, while efficient separation of structurally similar compounds is essential for accurate quantification. However, conventional LC methods are often time-intensive, limiting analytical throughput in large-scale and clinical applications. Here, we present rapid LC–trapped ion mobility spectrometry–tandem mass spectrometry (LC–TIMS–MS/MS) approaches that substantially reduce analysis time while preserving chromatographic resolution and quantitative reliability, with run times of around 2 minutes or below. Accelerated separations were achieved using shorter columns and elevated flow rates coupled to a trapped ion mobility mass spectrometer. The added ion mobility dimension improves molecular specificity and enhances confidence in compound annotation. To maximise metabolome coverage in a single chromatographic analysis, and to further increase throughput, we implemented automated derivatization of carboxylic acids, ketones, aldehydes, and amino acids using a liquid-handling platform, minimizing rate-limiting steps in sample preparation. The resulting workflow is well suited for high-throughput small-molecule analysis in large cohort studies and screening applications. The method was applied to the analysis of short-chain fatty acids, tricarboxylic acid cycle intermediates, and amino acids in human plasma and fecal samples.","dates":{"publication":"2026-06-30","submission":"2026-06-22"},"accession":"MTBLS14813","cross_references":{"MetaboLights":["MTBLC15366","MTBLC30768","MTBLC30772","MTBLC17418","MTBLC30776","MTBLC27570","MTBLC29016","MTBLC32664","MTBLC24996","MTBLC30769","MTBLC15741","MTBLC18012","MTBLC16810","MTBLC32816"],"ChEBI":["CHEBI:15366","CHEBI:30768","CHEBI:30772","CHEBI:17418","CHEBI:30776","CHEBI:27570","CHEBI:29016","CHEBI:32664","CHEBI:24996","CHEBI:30769","CHEBI:15741","CHEBI:18012","CHEBI:16810","CHEBI:32816"]}}