Feature identification and metabolite annotation for phenolics, caffeine, and caffeine metabolites were performed using MassLynx version 4.2 within the TargetLynx processing environment. Annotation was based on matching retention times and MRM transitions to authentic analytical standards, which served as the primary reference database. When standards were unavailable, identification was supported by diagnostic fragments reported in the literature and by the in house spectral library for phenolic compounds. TargetLynx handled peak detection, integration, and comparison to expected ion transitions. Peaks deviating by more than 0.05 min from reference retention times or lacking the expected fragmentation pattern were rejected to ensure high confidence annotation before quantification.
Feature identification and annotation were conducted in Xcalibur version 2.1. The software enabled manual and automated inspection of SRM or full scan MS2 chromatograms and comparison against authentic standards, which formed the primary reference database. Identification was accepted only when retention times matched reference values within 0.05 min and fragment ions aligned with expected transitions. Xcalibur facilitated the verification of spectral quality, extraction of ion chromatograms, and confirmation of molecular identity prior to quantitative analysis.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse-phase","Liquid Chromatography MS - positive - reverse-phase"],"chromatography_protocol":["Urine Phenolics, caffeine, and caffeine metabolites
Ultra-high-performance liquid chromatography (UHPLC) was carried out using an ACQUITY UHPLC I-Class Plus system (Waters Corporation, Milford, MA, USA) equipped with a binary pump, autosampler, and column oven. An ACQUITY Premier HSS T3 (1.8 µm, 2.1 x 100 mm; Waters) column with an ACQUITY Premier HSS T3 VanGuard precolumn (1.8 µm, 2.1 x 5 mm; Waters) was used. The injection volume was 5 µL. Samples were kept at 10 °C and the column at 40 °C. Mobile phase A consisted of water with 0.01% formic acid and mobile phase B consisted of acetonitrile with 0.01% formic acid. The gradient elution was as follows: 0 min, 99% A; 0.5 min, 99% A; 3.0 min, 85% A; 6.0 min, 50% A; 9.0 min, 5% A; 10.0 min, 5% A; 11.0 min, 99% A; 14.0 min, 99% A. The flow rate was 0.40 mL/min.
Creatinine
Creatinine analysis was performed using a UHPLC Accela 1250 system coupled to a linear ion trap mass spectrometer with a heated electrospray ionization source (Thermo Fisher Scientific). Separation was achieved on an XSelect HSS T3 (2.5 µm, 2.1 mm x 50 mm; Waters) column installed with a precolumn cartridge (Phenomenex, USA). The injection volume was 5 µL and the column temperature was maintained at 40 °C. Mobile phase A consisted of water containing 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid. Chromatographic separation was performed isocratically at 50% A and 50% B for 2 min at a flow rate of 0.20 mL/min.
Feces
Fecal samples were analyzed using a UHPLC Dionex Ultimate 3000 system coupled to a TSQ Vantage triple quadrupole mass spectrometer equipped with a heated electrospray ionization source (Thermo Fisher Scientific, San Jose, CA, USA). Separation was performed using a Kinetex Evo C18 (2.6 µm, 100 x 2.1 mm; Phenomenex) column installed with a Phenomenex precolumn cartridge. The mobile phases were acidified water with 0.01% formic acid (solvent A) and acidified acetonitrile with 0.01% formic acid (solvent B). The flow rate was 0.40 mL/min. The gradient program was: 0.0–0.5 min, 5% B; 0.5–7.5 min, linear increase to 40% B; 7.5–8.5 min, increase to 80% B; 8.5–10.5 min, hold at 80% B; 10.5–11.0 min, return to 5% B; 11.0–14.0 min, re-equilibration at 5% B (total run time: 14 min).
"],"publication":["Habitual Coffee Intake Shapes the Gut Microbiome and Modifies Host Physiology and Cognition."],"submitter_name":["Laila Zeraik"],"submitter_affiliation":["University of Parma"],"organism_part":["urine","feces"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["For phenolic analysis, urine samples were defrosted, vortexed, diluted in 0.1% formic acid in water (1:5 v/v), centrifuged at 17,968 × g for 5 min, and filtered (0.22 μm nylon filter). For caffeine and caffeine metabolites, a subsequent dilution in acidified water was performed (1:50 v/v). For creatinine, urine samples were centrifuged at 14.000 rpm for 5 min and subsequently diluted with water 0.1% formic acid (1:1000 v/v), vortexed, and transferred into vials for analysis.
Faecal samples were thawed, weighed, proportionally diluted in ethyl acetate acidified (1200 µL for every 300 mg of sample) with formic acid (0.1%), vortexed, and sonicated for 10 min at room temperature 29,30. Vortexing and sonication (5 min) were repeated once before centrifuging (14000 rpm, 10 min, 4 °C). The supernatant was recovered, and the pellet was re-extracted following the same procedure. The resulting supernatants were pooled, vortexed, dried in a vacuum concentrator, resuspended in 200 μL of MeOH: H2O (50:50, v/v acidified with formic acid (0.1%)), sonicated, centrifuged, and filtered (0.22 μm nylon) into vials for UHPLC-ESI-MS/MS analysis.
Raw data generated on the Waters UHPLC Xevo XS triple quadrupole system were acquired and processed using MassLynx version 4.2 with the TargetLynx application manager. MassLynx handled instrument control, chromatographic acquisition, and MRM signal recording. TargetLynx was used for automated peak detection, integration, and quantification.
Data from the Thermo Scientific systems were acquired and processed using Xcalibur version 2.1. Xcalibur controlled the UHPLC MS/MS platforms and recorded SRM or full scan MS2 chromatograms depending on the method. Raw files were processed within the software for peak inspection, fragment confirmation, and quantification based on calibration curves constructed from authentic standards or structurally similar compounds. Retention time matching and characteristic fragment ions were used to confirm compound identity, and peaks deviating by more than 0.05 min from reference retention times were excluded.
Biological samples were collected on the day of Visit 2 (V2), Visit 3 (V3) and Visit 4 (V4) and analysed as follows. The first urine of the day was collected and kept refrigerated in a sterile collection tube by each participant, and 4 ml of urine were aliquoted into 1ml of 0.25% sodium azide and stored at −80 °C. Participants were asked to collect the first freshly voided stool samples of the day in plastic containers containing an AnaeroGen sachet (Oxoid AGS AnaeroGen Compact, Fischer Scientific, Dublin). Stool samples were refrigerated using ice packs until delivery to the laboratory, where they were aliquoted and stored at −80 °C. Stool samples were collected the day of the visits as well as during the extra timepoints during the coffee washout and coffee intervention.
"],"omics_type":["Metabolomics"],"study_design":["Waters ACQUITY I-Class Sample Manager","Multi-omics study","Waters Xevo TQ-S","urine","untargeted analysis","Metabolome","Homo sapiens","Coffee","microbiome","experimental sample","caffeine","Thermo Scientific LTQ Orbitrap XL","Thermo Scientific Accela 1250 Pump","Thermo Scientific Dionex Ultimate 3000 UHPLC system","Thermo Scientific TSQ Vantage","feces"],"curator_keywords":["Waters ACQUITY I-Class Sample Manager","Multi-omics study","Waters Xevo TQ-S","urine","untargeted analysis","Metabolome","Homo sapiens","Coffee","microbiome","experimental sample","caffeine","Thermo Scientific LTQ Orbitrap XL","Thermo Scientific Accela 1250 Pump","Thermo Scientific Dionex Ultimate 3000 UHPLC system","Thermo Scientific TSQ Vantage","feces"],"mass_spectrometry_protocol":["Urine phenolics, caffeine, and caffeine metabolites
A Xevo XS triple quadrupole mass spectrometer with an electrospray ionization source (UHPLC ESI QqQ MS/MS, Waters) was used in both negative and positive ionization modes. The capillary voltage was set to 2.3 kV. The source temperature was set to 150 °C and the desolvation temperature to 600 °C. Gas flows were set as follows: cone gas 150 L/min, desolvation gas 800 L/min, and nebulizer gas 7 L/min. The mass spectrometer operated in multiple reaction monitoring (MRM) mode. Cone voltage and collision energy were optimized for each compound by infusion of reference standards. When standards were not available, energies were optimized during chromatographic analysis or inferred from structurally similar compounds. Instrument control and data acquisition were performed using MassLynx software version 4.2, and data processing was carried out using TargetLynx (Waters).
Creatinine
Mass spectrometric analysis was carried out using a system equipped with a HESI II ionization source. The capillary temperature was set to 275 °C and the source heater temperature to 300 °C. Sheath gas (nitrogen) was set to 40 arbitrary units, and auxiliary gas (nitrogen) to 5 arbitrary units. The source voltage was 4.5 kV. The capillary voltage and tube lens voltage were set to +20 V and +95 V, respectively. Ultra high purity helium was used as the collision gas, with a collision induced dissociation (CID) value of 55. Creatinine was analyzed using full scan MS2 in positive ionization mode, monitoring ions in the m/z range from 35 to 200. Chromatographic and spectral data were acquired using XCalibur software version 2.1 (Thermo Fisher Scientific).
Feces
Fecal extracts were analyzed using a mass spectrometer equipped with a HESI II ionization source. The capillary temperature was set to 275 °C and the source heater temperature to 250 °C. Sheath gas (nitrogen) was set to 40 arbitrary units, auxiliary gas to 5 arbitrary units, and sweep gas to 15 arbitrary units. The source voltage was 3 kV. The capillary voltage was set to -9 V and the tube lens voltage to -53 V. A total of 52 compounds were monitored in selected reaction monitoring (SRM) mode. Quantification was performed using calibration curves of available reference standards or, when standards were not available, by using structurally similar compounds. Data processing was performed using XCalibur software version 2.1.
"],"additional_accession":[]},"is_claimable":false,"name":"Habitual Coffee Intake Shapes the Gut Microbiome and Modifies Host Physiology and Cognition","description":"Coffee influences multiple physiological processes, including gut function, stress, cognition, and the microbiome. However, the mechanisms underlying these effects remain poorly understood. In this study, we examined coffee’s impact on the microbiota–gut–brain axis—a bidirectional communication pathway between the gut microbiome and the brain—and assessed whether these effects occur independently of caffeine in healthy participants. Significant group differences emerged in faecal microbiome composition, with coffee drinkers showing increased relative abundance of Cryptobacterium and Eggerthella species, alongside reduced levels of the metabolite’s indole-3-propionic acid, indole-3-carboxyaldehyde, and the neurotransmitter γ-aminobutyric acid. Behaviourally, coffee drinkers exhibited greater impulsivity and emotional reactivity, whereas non-coffee drinkers demonstrated better memory performance. Some alterations in the faecal metabolome were reversible following coffee abstinence, and reintroduction triggered acute microbiome changes independent of caffeine. An integrated model identified nine key metabolites—including theophylline, caffeine, and selected phenolic acids—strongly linked to microbial species and cognitive measures. These findings reveal previously unrecognised effects of coffee on the microbiota–gut–brain axis, suggesting that microbiome profiles could potentially predict coffee consumption patterns and highlighting a close association between coffee intake and gut microbial composition.
","dates":{"publication":"2026-04-07","submission":"2025-12-11"},"accession":"MTBLS13494","cross_references":{}}