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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/m_MTBLS14519_LC-MS_negative_reverse-phase-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/m_MTBLS14519_LC-MS_negative_reverse-phase-2_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/m_MTBLS14519_LC-MS_positive_reverse-phase-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/m_MTBLS14519_LC-MS_positive_reverse-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/s_MTBLS14519.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/a_MTBLS14519_LC-MS_positive_reverse-phase-2.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/a_MTBLS14519_LC-MS_positive_reverse-phase-3.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/a_MTBLS14519_LC-MS_negative_reverse-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519/a_MTBLS14519_LC-MS_negative_reverse-phase-3.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14519Metabolite identification was performed in Metaboscape 2025b using GNPS (https://www.nature.com/articles/nbt.3597) and MSDial (https://www.nature.com/articles/nmeth.3393).MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phaseExtracts were analyzed using the harmonized method established at Helmholtz Munich (HMGU). Chromatographic separation was performed on a Phenomenex Kinetex C18 column (100×2.1 mm, 1.7 µm) maintained at 40°C with a flow rate of 0.5 mL/min. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B).The linear gradient was programmed as follows: an initial hold at 5% B for 1.5 min, followed by a linear increase to 99.9% B over 8.5 min. This composition was held for 2.0 min before returning to initial conditions (5% B) at 12.1 min, with a final re-equilibration period of 2.9 min, totaling a 15-min run time. Regarding the in-house method, reverse phase separation was performed using an Acquity UPLC HSS-T3 (C18), 1.8 µm particle size, 2.1 × 100 mm column (Waters, Milford, MA, USA). The aqueous mobile phase was 0.1% formic acid in water (solvent A) and the organic one was 0.1% formic acid in methanol (solvent B). The mobile phase gradient was as follows (percentage of solvent B): 0% from 0 to 1.5 min, linearly increased to 10% from 1.5 to 4 min, linearly increased to 40% from 4 to 8 min, linearly increased to 100% from 8 to 12 min, and held at 100% from 12 to 14 min. Three minutes of equilibration in the initial conditions followed. The flow rate was kept at 0.35 mL/min. The injection volumes were 10 µL for the extraction optimization study and 20 µL for the BµS device and plasma comparison study.The HUMAN Ring Trial.Helen GikaAristotle University of Thessalonikireference compoundDried Blood SpotSolventmass spectrometry assayFrozen samples were thawed on ice, and the entire dried blood spot (20 μL) was transferred to a reaction tube. The spots were first extracted in 100 μL of water via sonication for 10 min in an ice-cooled bath, followed by centrifugation (14,000 rpm, 10 min, 0°C). After adding 400 μL of methanol, the samples were sonicated for an additional 10 min under the same conditions.The DBS cards were then removed, and the mixture was centrifuged (14,000 rpm, 10 min, 0°C). The resulting supernatant was divided into two aliquots for the respective chromatographic methods, with 50 μL from each sample pooled to create QC samples. All extracts were evaporated to dryness. Finally, samples were reconstituted in 200 μL of either ACN/H2O (20:80) for the harmonized method or MeOH/H2O (5:95) for the in-house method.reference compoundHomo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS14519Daniel Silva. Aristotle University of Thessaloniki. dmarque@auth.gr.Georgios Theodoridis. Aristotle University of Thessaloniki. Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.. gtheodor@auth.gr.Marlene Thaitumu. Aristotle University of Thessaloniki. School of Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece.. mthai@auth.gr.Helen Gika. Aristotle University of Thessaloniki. gkikae@auth.gr.Data processing was performed using the original data format on Metaboscape 2025b.Spiking stateChromatographic methodgkikae@auth.grDBS samples were prepared at the BIOMIC lab of Aristotle University of Thessaloniki. To evaluate the recovery and accurate annotation of biomarkers from biological specimens, venipuncture whole blood samples from 10 healthy male individuals were divided into two aliquots. One aliquot of the sample (200 μL) was spiked with a mixture of 12 endogenous metabolites (at various concentration levels for each individual) and a mixture of 8 exogenous compounds (at various concentration levels for each individual). Another aliquot (200 μL)  was kept unspiked. Twenty (20) μL of the samples were spotted on Whatman cards.Metabolomicsultra-performance liquid chromatography-mass spectrometryMetabolomicsDried Blood Spotuntargeted analysislong term referenceHomo sapiensring trial designdata-dependent acquisitionBruker Elute UHPLC systemsample preparation blankreference compound mixexperimental sampleelectrospray-ionisation quadrupole time-of-flight tandem mass spectrometryreference compoundBruker timsTOF Pro 2ion mobility separationSolventultra-performance liquid chromatography-mass spectrometryMetabolomicsDried Blood Spotuntargeted analysislong term referenceHomo sapiensring trial designdata-dependent acquisitionBruker Elute UHPLC systemsample preparation blankreference compound mixexperimental sampleelectrospray-ionisation quadrupole time-of-flight tandem mass spectrometryreference compoundBruker timsTOF Pro 2ion mobility separationSolventThe TIMS-qTOFMS was operated in data-dependent acquisition mode with a 20-to-1300 m/z scanning mass range. PASEF was performed in two ramps with a cycle time of 0.69 seconds. TIMS ramp was set with 1/k0 values going from 0.10 V.s/cm2 to 1.50 V.s/cm2 in 100 ms.The electrospray ionization (ESI) source was operated in positive and negative ion mode. For the positive mode, the following parameters were employed: end plate offset 500 V, capillary voltage 4500 V, nebulizer gas pressure 2.2 bars, drying gas flow rate 10 L/min, temperature 220 C, and quadrupole energy 5.0 eV. For the negative mode, end plate offset was 500 V, capillary voltage 3600 V, nebulizer gas pressure 2.2 bars, drying gas flow rate 10 L/min, temperature 220 C, and quadrupole energy 5.0 eV.The collision-induced decay energy was set for each mass range. Sodium formate solution (10 mM) and AgilentTM calibrant mix were used for calibration by direct infusion into the MS at a 10 μL/h flow rate for the first 0.5 min of every injection.L-phenylalanine1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydro-2-benzofuran-5-carbonitrile(R)-carnitine4-QUINOLINECARBOXYLICACIDMethadoneADPAnthranilicacidcaffeineN(2)-phenylacetyl-L-glutamineUnknownhypoxanthine(5-hydroxyindol-3-yl)acetic acidethylenediaminetetraacetic acidPesticide3_Carbofuran_C12H15NO3_Furadan2-NaphthylamineN,N-dimethyl-beta-alaninebenzocaineindole-3-carbaldehydeKYNURENICACIDadenosine 5'-monophosphate5-HYDROXYINDOLEACETATEO-propanoyl-L-carnitinecreatineO-acetyl-L-carnitine1-hexadecanoyl-sn-glycero-3-phosphocholineL-isoleucinefalseMethod Comparison using DBS samplesMetabolomics serves as a critical pillar in clinical research, yet the lack of standardized protocols often limits the comparability of data across different platforms and institutions. The HUMAN (Harmonizing and Unifying Blood Metabolomics Network) has as one of its goals to to advance the field of metabolomics by performing cross-laboratory comparisons, integration and co-evaluation of data analysis tools. The present Ring Trial was established as a multi-center collaborative effort to address these inconsistencies by systematically evaluating the impact of varying analytical setups on metabolite detection and identification. While the broader initiative encompasses multiple phases of standardization, the present work focuses specifically on the Phase 2 of the ring trial, which evaluates the analytical robustness of Dried Blood Spot (DBS) analysis.In this phase, whole blood samples from 10 healthy individuals were used to create a controlled 'artificial metabolic disruption' model. Samples were divided into two cohorts: one unspiked and one spiked with a blinded mixture of 12 endogenous metabolites and 8 exogenous compounds at varying concentrations. These samples were spotted onto Whatman cards to simulate real-world clinical specimens. Participating laboratories analyzed the blinded samples using both a common harmonized LC-MS method and their own lab-specific protocols to assess the recovery and accurate annotation of these 'artificial biomarkers.' By comparing results across diverse mass spectrometry platforms, this study quantifies the efficiency of different workflows in identifying unknown compounds in complex matrices. The current report is dedicated exclusively to the methodology and outcomes of this second phase analyzed at the BIOMIC laboratory at Aristotle University of Thessaloniki.2026-05-192026-05-19MTBLS14519