#METABOLOMICS WORKBENCH diegogr_20251021_073850 DATATRACK_ID:6577 STUDY_ID:ST004304 ANALYSIS_ID:AN007161 PROJECT_ID:PR002720 VERSION 1 CREATED_ON October 22, 2025, 9:42 am #PROJECT PR:PROJECT_TITLE Untargeted UHPLC-Q-Orbitrap Metabolomic Profiling of Cattle Serum after PR:PROJECT_TITLE Testosterone Propionate Administration PR:PROJECT_TYPE Untargeted Metabolomics PR:PROJECT_SUMMARY The misuse of anabolic androgenic steroids in livestock remains a major concern PR:PROJECT_SUMMARY for food safety and regulatory monitoring, as exogenous administration of PR:PROJECT_SUMMARY endogenous hormones such as testosterone is difficult to confirm using PR:PROJECT_SUMMARY conventional analytical approaches. This project aimed to develop an untargeted PR:PROJECT_SUMMARY metabolomics workflow based on ultra-high-performance liquid chromatography PR:PROJECT_SUMMARY coupled to high-resolution Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) to PR:PROJECT_SUMMARY investigate metabolic alterations in cattle serum following testosterone PR:PROJECT_SUMMARY propionate administration. The study focused on optimizing data acquisition and PR:PROJECT_SUMMARY processing strategies, including feature detection, alignment, and statistical PR:PROJECT_SUMMARY evaluation using multivariate and univariate approaches. The resulting workflow PR:PROJECT_SUMMARY provides a comprehensive analytical framework that can support future regulatory PR:PROJECT_SUMMARY and research applications related to hormone misuse detection in food-producing PR:PROJECT_SUMMARY animals. PR:INSTITUTE Universidade Federal de Minas Gerais PR:DEPARTMENT Department of Chemistry PR:LABORATORY 261A PR:LAST_NAME Rocha PR:FIRST_NAME Diego PR:ADDRESS Av Antônio Carlos 6627, Belo Horizonte, MG, 31270-901, Brazil PR:EMAIL diegogr.ufmg@gmail.com PR:PHONE 553134095734 #STUDY ST:STUDY_TITLE Untargeted UHPLC-Q-Orbitrap Metabolomic Profiling of Cattle Serum after ST:STUDY_TITLE Testosterone Propionate Administration ST:STUDY_TYPE Untargeted Metabolomics ST:STUDY_SUMMARY This study presents an untargeted metabolomic analysis of cattle serum using ST:STUDY_SUMMARY UHPLC-Q-Orbitrap mass spectrometry to investigate metabolic alterations ST:STUDY_SUMMARY following testosterone propionate administration. The data include raw and ST:STUDY_SUMMARY processed MS files, feature tables, and statistical analyses supporting ST:STUDY_SUMMARY biomarker discovery and pathway elucidation. ST:INSTITUTE Universidade Federal de Minas Gerais ST:DEPARTMENT Department of Chemistry ST:LABORATORY 261A ST:LAST_NAME Rocha ST:FIRST_NAME Diego ST:ADDRESS Av. Antônio Carlos 6627, Belo Horizonte, MG, 31270-901, Brazil ST:EMAIL diegogr.ufmg@gmail.com ST:PHONE 553134095734 ST:NUM_GROUPS - ST:TOTAL_SUBJECTS - ST:NUM_MALES - ST:NUM_FEMALES - ST:STUDY_COMMENTS - ST:PUBLICATIONS - ST:DISEASE_ASSOCIATION - #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Bos taurus SU:TAXONOMY_ID 9913 SU:GENOTYPE_STRAIN - SU:AGE_OR_AGE_RANGE - SU:WEIGHT_OR_WEIGHT_RANGE - SU:HEIGHT_OR_HEIGHT_RANGE - SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER - SU:ANIMAL_HOUSING - SU:ANIMAL_LIGHT_CYCLE - SU:ANIMAL_FEED - SU:ANIMAL_WATER - SU:ANIMAL_INCLUSION_CRITERIA - SU:SPECIES_GROUP - #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - T1-D1 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T1-D1.mzML SUBJECT_SAMPLE_FACTORS - T1-D2 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T1-D2.mzML SUBJECT_SAMPLE_FACTORS - T1-D5 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T1-D5.mzML SUBJECT_SAMPLE_FACTORS - T1-D8 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T1-D8.mzML SUBJECT_SAMPLE_FACTORS - T2-D1 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T2-D1.mzML SUBJECT_SAMPLE_FACTORS - T2-D2 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T2-D2.mzML SUBJECT_SAMPLE_FACTORS - T2-D5 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T2-D5.mzML SUBJECT_SAMPLE_FACTORS - T2-D8 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T2-D8.mzML SUBJECT_SAMPLE_FACTORS - T3-D1 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T3-D1.mzML SUBJECT_SAMPLE_FACTORS - T3-D2 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T3-D2.mzML SUBJECT_SAMPLE_FACTORS - T3-D5 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T3-D5.mzML SUBJECT_SAMPLE_FACTORS - T3-D8 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T3-D8.mzML SUBJECT_SAMPLE_FACTORS - T4-D1 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T4-D1.mzML SUBJECT_SAMPLE_FACTORS - T4-D2 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T4-D2.mzML SUBJECT_SAMPLE_FACTORS - T4-D5 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T4-D5.mzML SUBJECT_SAMPLE_FACTORS - T4-D8 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T4-D8.mzML SUBJECT_SAMPLE_FACTORS - T1-D12 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T1-D12.mzML SUBJECT_SAMPLE_FACTORS - T1-D15 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T1-D15.mzML SUBJECT_SAMPLE_FACTORS - T1-D18 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T1-D18.mzML SUBJECT_SAMPLE_FACTORS - T2-D12 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T2-D12.mzML SUBJECT_SAMPLE_FACTORS - T2-D15 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T2-D15.mzML SUBJECT_SAMPLE_FACTORS - T2-D18 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T2-D18.mzML SUBJECT_SAMPLE_FACTORS - T3-D12 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T3-D12.mzML SUBJECT_SAMPLE_FACTORS - T3-D15 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T3-D15.mzML SUBJECT_SAMPLE_FACTORS - T3-D18 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T3-D18.mzML SUBJECT_SAMPLE_FACTORS - T4-D12 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T4-D12.mzML SUBJECT_SAMPLE_FACTORS - T4-D15 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T4-D15.mzML SUBJECT_SAMPLE_FACTORS - T4-D18 Sample source:Serum | Treated:Treated RAW_FILE_NAME(T1-D1.mzML)=T4-D18.mzML SUBJECT_SAMPLE_FACTORS - T1-D-5 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T1-D-5.mzML SUBJECT_SAMPLE_FACTORS - T1-D-4 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T1-D-4.mzML SUBJECT_SAMPLE_FACTORS - T1-D0 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T1-D0.mzML SUBJECT_SAMPLE_FACTORS - T2-D-5 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T2-D-5.mzML SUBJECT_SAMPLE_FACTORS - T2-D-4 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T2-D-4.mzML SUBJECT_SAMPLE_FACTORS - T2-D0 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T2-D0.mzML SUBJECT_SAMPLE_FACTORS - T3-D-5 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T3-D-5.mzML SUBJECT_SAMPLE_FACTORS - T3-D-4 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T3-D-4.mzML SUBJECT_SAMPLE_FACTORS - T3-D0 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T3-D0.mzML SUBJECT_SAMPLE_FACTORS - T4-D-5 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T4-D-5.mzML SUBJECT_SAMPLE_FACTORS - T4-D-4 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T4-D-4.mzML SUBJECT_SAMPLE_FACTORS - T4-D0 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=T4-D0.mzML SUBJECT_SAMPLE_FACTORS - C1-D1 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C1-D1.mzML SUBJECT_SAMPLE_FACTORS - C1-D8 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C1-D8.mzML SUBJECT_SAMPLE_FACTORS - C1-D18 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C1-D18.mzML SUBJECT_SAMPLE_FACTORS - C2-D1 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C2-D1.mzML SUBJECT_SAMPLE_FACTORS - C2-D8 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C2-D8.mzML SUBJECT_SAMPLE_FACTORS - C2-D18 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C2-D18.mzML SUBJECT_SAMPLE_FACTORS - C3-D1 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C3-D1.mzML SUBJECT_SAMPLE_FACTORS - C3-D8 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C3-D8.mzML SUBJECT_SAMPLE_FACTORS - C3-D18 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C3-D18.mzML SUBJECT_SAMPLE_FACTORS - C4-D1 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C4-D1.mzML SUBJECT_SAMPLE_FACTORS - C4-D8 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C4-D8.mzML SUBJECT_SAMPLE_FACTORS - C4-D18 Sample source:Serum | Treated:Control RAW_FILE_NAME(T1-D1.mzML)=C4-D18.mzML SUBJECT_SAMPLE_FACTORS - QC-D2-A Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D2-A.mzML SUBJECT_SAMPLE_FACTORS - QC-D2-B Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D2-B.mzML SUBJECT_SAMPLE_FACTORS - QC-D2-C Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D2-C.mzML SUBJECT_SAMPLE_FACTORS - QC-D2-D Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D2-D.mzML SUBJECT_SAMPLE_FACTORS - QC-D5-A Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D5-A.mzML SUBJECT_SAMPLE_FACTORS - QC-D5-B Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D5-B.mzML SUBJECT_SAMPLE_FACTORS - QC-D5-C Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D5-C.mzML SUBJECT_SAMPLE_FACTORS - QC-D5-D Sample source:Serum | Treated:QC RAW_FILE_NAME(T1-D1.mzML)=QC-D5-D.mzML #COLLECTION CO:COLLECTION_SUMMARY Blood samples were collected from male cattle by jugular venipuncture. Serum was CO:COLLECTION_SUMMARY obtained after clotting and centrifugation, and stored at −80 °C until CO:COLLECTION_SUMMARY analysis. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY Serum samples (n = 52) were collected from two groups of animals: four treated TR:TREATMENT_SUMMARY steers and four untreated controls. For the treated group (n = 40 samples), TR:TREATMENT_SUMMARY collections were performed on days −5, −4, 0 (before drug administration), 1 TR:TREATMENT_SUMMARY (6 h after administration), 2, 5, 8, 12, 15, and 18. For the control group (n = TR:TREATMENT_SUMMARY 12 samples), collections were performed on days 1, 8, and 18. In addition, TR:TREATMENT_SUMMARY pre-treatment samples from the treated steers (n = 12 from days −5, −4, 0) TR:TREATMENT_SUMMARY were included as controls. For supervised analysis, samples from days 2, 5, and TR:TREATMENT_SUMMARY 8 of the treated group were considered truly positive, while all samples from TR:TREATMENT_SUMMARY untreated animals and pre-treatment samples from treated animals were considered TR:TREATMENT_SUMMARY truly negative (n = 24). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum samples (500 µL) were firstly spiked with 100 µL of the internal SP:SAMPLEPREP_SUMMARY standard (IS - 17β-boldenone-d3) solution in Eppendorf tubes (2 mL), followed SP:SAMPLEPREP_SUMMARY by the addition of 50 mg of NaCl and 1 mL of acetonitrile. The tubes were SP:SAMPLEPREP_SUMMARY vortexed for 30 s and then two quality control (QC) samples were centrifuged at SP:SAMPLEPREP_SUMMARY 17,970 g at 0 °C for 30 min using a Sigma 3-30KS high-speed centrifuge (Sigma SP:SAMPLEPREP_SUMMARY Laborzentrifugen GmbH, Osterode am Harz, Germany). The supernatant (500 µL) was SP:SAMPLEPREP_SUMMARY transferred to 2 mL autosampler vials, to which 300 µL of ultrapure water were SP:SAMPLEPREP_SUMMARY added. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Accela 1250 CH:COLUMN_NAME Thermo Hypersil GOLD aQ (100 x 2.1mm,1.9um) CH:SOLVENT_A Water CH:SOLVENT_B Methanol CH:FLOW_GRADIENT 5% B for 0.8 min; linear increase to 100% B until 10.0 min; 100% B until 14.0 CH:FLOW_GRADIENT min; linear decrease to 5% B until 17.0 min; 5% B until 24.0 min. CH:FLOW_RATE 0.3 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Serum samples were analyzed using a Q-Exactive™ Orbitrap mass spectrometer MS:MS_COMMENTS (Thermo Scientific, Bremen, Germany) equipped with a heated electrospray MS:MS_COMMENTS ionization (HESI) source operated in positive ion mode. Full-scan (FS) and MS:MS_COMMENTS all-ion fragmentation (AIF) data were acquired over an m/z range of 70–600. MS:MS_COMMENTS The resolving power was 70,000 FWHM at m/z 200 for FS and 17,500 FWHM for AIF. MS:MS_COMMENTS The spray voltage was set to 4.0 kV, capillary temperature to 380 °C, and MS:MS_COMMENTS sheath and auxiliary gas (nitrogen) pressures to 15 and 44 arbitrary units, MS:MS_COMMENTS respectively. The automatic gain control (AGC) targets were 3 × 10⁶ ions (FS) MS:MS_COMMENTS and 2 × 10⁵ ions (AIF), with a maximum injection time of 200 ms. Data MS:MS_COMMENTS acquisition and control were performed using Xcalibur software (v4.2.47, Thermo MS:MS_COMMENTS Fisher Scientific). MS:MS_RESULTS_FILE ST004304_AN007161_Results.txt UNITS:arbitrary units Has m/z:Yes Has RT:Yes RT units:Minutes #END