#METABOLOMICS WORKBENCH SIMR_Core_Facility_20231108_225357 DATATRACK_ID:4449 STUDY_ID:ST002975 ANALYSIS_ID:AN004885 PROJECT_ID:PR001852 VERSION 1 CREATED_ON November 15, 2023, 12:35 pm #PROJECT PR:PROJECT_TITLE Metabolomics Insights into Doxorubicin and 5-Fluorouracil Combination Therapy in PR:PROJECT_TITLE Triple-Negative Breast Cancer: A Xenograft Model Study PR:PROJECT_TYPE LC-MS/MS PR:PROJECT_SUMMARY Background: Breast cancer is one of the most prevalent malignancies and a PR:PROJECT_SUMMARY leading cause of death among women worldwide. Among its subtypes, PR:PROJECT_SUMMARY triple-negative breast cancer (TNBC), which poses significant clinical PR:PROJECT_SUMMARY challenges due to its aggressive behavior and limited treatment options. Aim: PR:PROJECT_SUMMARY This study explored the effects of doxorubicin (DOX) and 5-fluorouracil (5-FU) PR:PROJECT_SUMMARY as monotherapies and in combination on MDA-MB-231 xenograft model. Employing PR:PROJECT_SUMMARY advanced metabolomics analysis, the study was designed to investigate molecular PR:PROJECT_SUMMARY alterations triggered by these treatments. Methods: State-of-the-art PR:PROJECT_SUMMARY metabolomics analysis using Ultra-high-performance liquid PR:PROJECT_SUMMARY chromatography-electrospray ionization quadrupole time-of-flight mass PR:PROJECT_SUMMARY spectrometry (UHPLC-ESI-QTOF-MS) was conducted including comprehensive plasma PR:PROJECT_SUMMARY and tumor tissue sample profiling. Results: The study explored alterations PR:PROJECT_SUMMARY induced by DOX, 5-FU, and their combination treatment. Each treatment group PR:PROJECT_SUMMARY exhibited unique metabolic profiles in plasma and tumor analysis. Univariate and PR:PROJECT_SUMMARY enrichment analyses identified alterations in metabolic pathways, including PR:PROJECT_SUMMARY glycine and serine metabolism, spermidine and spermine biosynthesis, and purine PR:PROJECT_SUMMARY and pyrimidine pathways. The combination of DOX and 5-FU significantly PR:PROJECT_SUMMARY influenced plasma and tumor metabolites. The comprehensive metabolic profiling PR:PROJECT_SUMMARY of both plasma and tumor samples shed light on the intricate changes within the PR:PROJECT_SUMMARY tumor microenvironment and their systemic implications. Conclusion: The study PR:PROJECT_SUMMARY findings offer insights into the metabolic vulnerabilities of TNBC in vivo PR:PROJECT_SUMMARY induced by the studied chemotherapeutics. These findings highlight the involved PR:PROJECT_SUMMARY metabolites and metabolic pathways in the response of MDA-MB-231 cells to DOX, PR:PROJECT_SUMMARY 5-FU, and their combination which advance our understanding of TNBC treatment PR:PROJECT_SUMMARY strategies, offering new possibilities for enhancing therapeutic outcomes. PR:INSTITUTE Sharjah Institute for Medical Research PR:LAST_NAME Facility PR:FIRST_NAME Core PR:ADDRESS M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, PR:ADDRESS UAE, Sharjah, 000, United Arab Emirates PR:EMAIL tims-tof@sharjah.ac.ae PR:PHONE +971 6 5057656 #STUDY ST:STUDY_TITLE Metabolomics Insights into Doxorubicin and 5-Fluorouracil Combination Therapy in ST:STUDY_TITLE Triple-Negative Breast Cancer: A Xenograft Model Study (Part 1) ST:STUDY_SUMMARY Background: Breast cancer is one of the most prevalent malignancies and a ST:STUDY_SUMMARY leading cause of death among women worldwide. Among its subtypes, ST:STUDY_SUMMARY triple-negative breast cancer (TNBC), which poses significant clinical ST:STUDY_SUMMARY challenges due to its aggressive behavior and limited treatment options. Aim: ST:STUDY_SUMMARY This study explored the effects of doxorubicin (DOX) and 5-fluorouracil (5-FU) ST:STUDY_SUMMARY as monotherapies and in combination on MDA-MB-231 xenograft model. Employing ST:STUDY_SUMMARY advanced metabolomics analysis, the study was designed to investigate molecular ST:STUDY_SUMMARY alterations triggered by these treatments. Methods: State-of-the-art ST:STUDY_SUMMARY metabolomics analysis using Ultra-high-performance liquid ST:STUDY_SUMMARY chromatography-electrospray ionization quadrupole time-of-flight mass ST:STUDY_SUMMARY spectrometry (UHPLC-ESI-QTOF-MS) was conducted including comprehensive plasma ST:STUDY_SUMMARY and tumor tissue sample profiling. Results: The study explored alterations ST:STUDY_SUMMARY induced by DOX, 5-FU, and their combination treatment. Each treatment group ST:STUDY_SUMMARY exhibited unique metabolic profiles in plasma and tumor analysis. Univariate and ST:STUDY_SUMMARY enrichment analyses identified alterations in metabolic pathways, including ST:STUDY_SUMMARY glycine and serine metabolism, spermidine and spermine biosynthesis, and purine ST:STUDY_SUMMARY and pyrimidine pathways. The combination of DOX and 5-FU significantly ST:STUDY_SUMMARY influenced plasma and tumor metabolites. The comprehensive metabolic profiling ST:STUDY_SUMMARY of both plasma and tumor samples shed light on the intricate changes within the ST:STUDY_SUMMARY tumor microenvironment and their systemic implications. Conclusion: The study ST:STUDY_SUMMARY findings offer insights into the metabolic vulnerabilities of TNBC in vivo ST:STUDY_SUMMARY induced by the studied chemotherapeutics. These findings highlight the involved ST:STUDY_SUMMARY metabolites and metabolic pathways in the response of MDA-MB-231 cells to DOX, ST:STUDY_SUMMARY 5-FU, and their combination which advance our understanding of TNBC treatment ST:STUDY_SUMMARY strategies, offering new possibilities for enhancing therapeutic outcomes. ST:INSTITUTE Sharjah Institute for Medical Research ST:LAST_NAME Facility ST:FIRST_NAME Core ST:ADDRESS M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, ST:ADDRESS UAE, Sharjah, 000, United Arab Emirates ST:EMAIL tims-tof@sharjah.ac.ae ST:STUDY_TYPE LC/MS/MS ST:PHONE +971 6 5057656 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES BALB/C nude mice #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 - 1A1-01-10368 Treatment:Doxorubicin RAW_FILE_NAME=1A1-01-10368.d SUBJECT_SAMPLE_FACTORS - 1A1-02-10369 Treatment:Doxorubicin RAW_FILE_NAME=1A1-02-10369.d SUBJECT_SAMPLE_FACTORS - 1A2-01-10370 Treatment:Doxorubicin RAW_FILE_NAME=1A2-01-10370.d SUBJECT_SAMPLE_FACTORS - 1A2-02-10371 Treatment:Doxorubicin RAW_FILE_NAME=1A2-02-10371.d SUBJECT_SAMPLE_FACTORS - 1A3-01-10372 Treatment:Doxorubicin RAW_FILE_NAME=1A3-01-10372.d SUBJECT_SAMPLE_FACTORS - 1A3-02-10373 Treatment:Doxorubicin RAW_FILE_NAME=1A3-02-10373.d SUBJECT_SAMPLE_FACTORS - 1B1-01-10374 Treatment:Doxorubicin RAW_FILE_NAME=1B1-01-10374.d SUBJECT_SAMPLE_FACTORS - 1B1-02-10375 Treatment:Doxorubicin RAW_FILE_NAME=1B1-02-10375.d SUBJECT_SAMPLE_FACTORS - 1B2-01-10376 Treatment:Doxorubicin RAW_FILE_NAME=1B2-01-10376.d SUBJECT_SAMPLE_FACTORS - 1B2-02-10377 Treatment:Doxorubicin RAW_FILE_NAME=1B2-02-10377.d SUBJECT_SAMPLE_FACTORS - 1B3-01-10378 Treatment:Doxorubicin RAW_FILE_NAME=1B3-01-10378.d SUBJECT_SAMPLE_FACTORS - 1B3-02-10379 Treatment:Doxorubicin RAW_FILE_NAME=1B3-02-10379.d SUBJECT_SAMPLE_FACTORS - 2A1-01-10381 Treatment:5-fluorouracil RAW_FILE_NAME=2A1-01-10381.d SUBJECT_SAMPLE_FACTORS - 2A1-02-10382 Treatment:5-fluorouracil RAW_FILE_NAME=2A1-02-10382.d SUBJECT_SAMPLE_FACTORS - 2A2-01-10383 Treatment:5-fluorouracil RAW_FILE_NAME=2A2-01-10383.d SUBJECT_SAMPLE_FACTORS - 2A2-02-10384 Treatment:5-fluorouracil RAW_FILE_NAME=2A2-02-10384.d SUBJECT_SAMPLE_FACTORS - 2B1-01-10385 Treatment:5-fluorouracil RAW_FILE_NAME=2B1-01-10385.d SUBJECT_SAMPLE_FACTORS - 2B1-02-10386 Treatment:5-fluorouracil RAW_FILE_NAME=2B1-02-10386.d SUBJECT_SAMPLE_FACTORS - 2B2-01-10387 Treatment:5-fluorouracil RAW_FILE_NAME=2B2-01-10387.d SUBJECT_SAMPLE_FACTORS - 2B2-02-10388 Treatment:5-fluorouracil RAW_FILE_NAME=2B2-02-10388.d SUBJECT_SAMPLE_FACTORS - 2B3-01-10389 Treatment:5-fluorouracil RAW_FILE_NAME=2B3-01-10389.d SUBJECT_SAMPLE_FACTORS - 2B3-02-10390 Treatment:5-fluorouracil RAW_FILE_NAME=2B3-02-10390.d SUBJECT_SAMPLE_FACTORS - 3A1-01-10392 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3A1-01-10392.d SUBJECT_SAMPLE_FACTORS - 3A1-02-10393 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3A1-02-10393.d SUBJECT_SAMPLE_FACTORS - 3A2-01-10394 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3A2-01-10394.d SUBJECT_SAMPLE_FACTORS - 3A2-02-10395 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3A2-02-10395.d SUBJECT_SAMPLE_FACTORS - 3B1-01-10396 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3B1-01-10396.d SUBJECT_SAMPLE_FACTORS - 3B1-02-10397 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3B1-02-10397.d SUBJECT_SAMPLE_FACTORS - 3B2-01-10398 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3B2-01-10398.d SUBJECT_SAMPLE_FACTORS - 3B2-02-10399 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3B2-02-10399.d SUBJECT_SAMPLE_FACTORS - 3B3-01-10400 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3B3-01-10400.d SUBJECT_SAMPLE_FACTORS - 3B3-02-10401 Treatment:Doxorubicin + 5-fluorouracil RAW_FILE_NAME=3B3-02-10401.d SUBJECT_SAMPLE_FACTORS - 4A1-01-10403 Treatment:Positive Control RAW_FILE_NAME=4A1-01-10403.d SUBJECT_SAMPLE_FACTORS - 4A1-02-10404 Treatment:Positive Control RAW_FILE_NAME=4A1-02-10404.d SUBJECT_SAMPLE_FACTORS - 4A2-01-10405 Treatment:Positive Control RAW_FILE_NAME=4A2-01-10405.d SUBJECT_SAMPLE_FACTORS - 4A2-02-10406 Treatment:Positive Control RAW_FILE_NAME=4A2-02-10406.d SUBJECT_SAMPLE_FACTORS - 4A4-01-10407 Treatment:Positive Control RAW_FILE_NAME=4A4-01-10407.d SUBJECT_SAMPLE_FACTORS - 4A4-02-10408 Treatment:Positive Control RAW_FILE_NAME=4A4-02-10408.d SUBJECT_SAMPLE_FACTORS - 4B1-01-10409 Treatment:Positive Control RAW_FILE_NAME=4B1-01-10409.d SUBJECT_SAMPLE_FACTORS - 4B1-02-10410 Treatment:Positive Control RAW_FILE_NAME=4B1-02-10410.d SUBJECT_SAMPLE_FACTORS - 4B2-01-10411 Treatment:Positive Control RAW_FILE_NAME=4B2-01-10411.d SUBJECT_SAMPLE_FACTORS - 4B2-02-10412 Treatment:Positive Control RAW_FILE_NAME=4B2-02-10412.d SUBJECT_SAMPLE_FACTORS - 4B3-01-10413 Treatment:Positive Control RAW_FILE_NAME=4B3-01-10413.d SUBJECT_SAMPLE_FACTORS - 4B3-02-10414 Treatment:Positive Control RAW_FILE_NAME=4B3-02-10414.d SUBJECT_SAMPLE_FACTORS - 5A1-01-10416 Treatment:Negative Control RAW_FILE_NAME=5A1-01-10416.d SUBJECT_SAMPLE_FACTORS - 5A1-02-10417 Treatment:Negative Control RAW_FILE_NAME=5A1-02-10417.d SUBJECT_SAMPLE_FACTORS - 5A2-01-10418 Treatment:Negative Control RAW_FILE_NAME=5A2-01-10418.d SUBJECT_SAMPLE_FACTORS - 5A2-02-10419 Treatment:Negative Control RAW_FILE_NAME=5A2-02-10419.d SUBJECT_SAMPLE_FACTORS - 5A3-01-10420 Treatment:Negative Control RAW_FILE_NAME=5A3-01-10420.d SUBJECT_SAMPLE_FACTORS - 5A3-02-10421 Treatment:Negative Control RAW_FILE_NAME=5A3-02-10421.d SUBJECT_SAMPLE_FACTORS - 5B1-01-10422 Treatment:Negative Control RAW_FILE_NAME=5B1-01-10422.d SUBJECT_SAMPLE_FACTORS - 5B1-02-10423 Treatment:Negative Control RAW_FILE_NAME=5B1-02-10423.d SUBJECT_SAMPLE_FACTORS - 5B2-01-10424 Treatment:Negative Control RAW_FILE_NAME=5B2-01-10424.d SUBJECT_SAMPLE_FACTORS - 5B2-02-10425 Treatment:Negative Control RAW_FILE_NAME=5B2-02-10425.d SUBJECT_SAMPLE_FACTORS - 5B3-01-10426 Treatment:Negative Control RAW_FILE_NAME=5B3-01-10426.d SUBJECT_SAMPLE_FACTORS - 5B3-02-10427 Treatment:Negative Control RAW_FILE_NAME=5B3-02-10427.d #COLLECTION CO:COLLECTION_SUMMARY MDA-MB-231 cell line was cultured in DMEM medium supplemented with 10% fetal CO:COLLECTION_SUMMARY bovine serum and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA) CO:COLLECTION_SUMMARY at 37 °C in a humidified atmosphere containing 5% CO2. This study was approved CO:COLLECTION_SUMMARY by the Institutional Animal Care and Use Committee (IACUC) at the University of CO:COLLECTION_SUMMARY Sharjah (Ethical Approval Number: ACUC-06-08-2022). Standard ethical guidelines CO:COLLECTION_SUMMARY were followed in all animal procedures conducted. Thirty female BALB/C nude mice CO:COLLECTION_SUMMARY (8-10 weeks age), with an average weight of 26-27 g, were included in the study CO:COLLECTION_SUMMARY and maintained in our institutional animal facility in accordance with following CO:COLLECTION_SUMMARY the animal care guidelines. The mice were randomized into five experimental CO:COLLECTION_SUMMARY groups (6 per group). Twenty-four mice were selected for the (CDX) model, and CO:COLLECTION_SUMMARY one group served as the negative control that didn’t receive any treatment. CO:COLLECTION_SUMMARY MDA-MB-231 cells (2 × 106 in 50 μL PBS & 50 μL Matrigel) were injected CO:COLLECTION_SUMMARY subcutaneously into the neck region of the mice. The mice were allowed to reach CO:COLLECTION_SUMMARY a tumor volume of 150-200 mm3 before further treatment. Then, tumor-bearing mice CO:COLLECTION_SUMMARY were randomized into the following groups: untreated xenografts (positive CO:COLLECTION_SUMMARY controls) and three treatment groups, DOX, 5-FU, and a combination of DOX and CO:COLLECTION_SUMMARY 5-FU. The mice in the DOX group were administered 1mg/kg of DOX once weekly , CO:COLLECTION_SUMMARY and the mice in the 5-FU group received 50 mg/kg of 5-FU daily for five CO:COLLECTION_SUMMARY consecutive days both treatments administered via an intraperitoneal (i.p.) CO:COLLECTION_SUMMARY route. The mice in the combination therapy group received DOX & 5-FU as separate CO:COLLECTION_SUMMARY injections, and the treatment duration was two weeks. An overview of the CO:COLLECTION_SUMMARY experiment flow. The body weight of the mice was measured at the start of the CO:COLLECTION_SUMMARY study and twice weekly from the beginning of the treatments. Tumor growth and CO:COLLECTION_SUMMARY progression were monitored by palpation and measurement of tumor size CO:COLLECTION_SUMMARY periodically using a digital vernier caliper. The tumor volume in cm3 was CO:COLLECTION_SUMMARY determined using the formula (volume = π/6 × length × width2). All mice were CO:COLLECTION_SUMMARY anesthetized and euthanized via cardiac puncture at the end of the treatment CO:COLLECTION_SUMMARY period. Tumors were excised and weighed, and their sizes were measured. Also, CO:COLLECTION_SUMMARY blood serum was collected from each mouse. Both tumor and serum samples were CO:COLLECTION_SUMMARY stored at -80°C for further analysis. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Then, tumor-bearing mice were randomized into the following groups: untreated TR:TREATMENT_SUMMARY xenografts (positive controls) and three treatment groups, DOX, 5-FU, and a TR:TREATMENT_SUMMARY combination of DOX and 5-FU. The mice in the DOX group were administered 1mg/kg TR:TREATMENT_SUMMARY of DOX once weekly , and the mice in the 5-FU group received 50 mg/kg of 5-FU TR:TREATMENT_SUMMARY daily for five consecutive days both treatments administered via an TR:TREATMENT_SUMMARY intraperitoneal (i.p.) route [32]. The mice in the combination therapy group TR:TREATMENT_SUMMARY received DOX & 5-FU as separate injections, and the treatment duration was two TR:TREATMENT_SUMMARY weeks. An overview of the experiment flow. The body weight of the mice was TR:TREATMENT_SUMMARY measured at the start of the study and twice weekly from the beginning of the TR:TREATMENT_SUMMARY treatments. Tumor growth and progression were monitored by palpation and TR:TREATMENT_SUMMARY measurement of tumor size periodically using a digital vernier caliper. The TR:TREATMENT_SUMMARY tumor volume in cm3 was determined using the formula (volume = π/6 × length × TR:TREATMENT_SUMMARY width2) [33]. All mice were anesthetized and euthanized via cardiac puncture at TR:TREATMENT_SUMMARY the end of the treatment period. Tumors were excised and weighed, and their TR:TREATMENT_SUMMARY sizes were measured. Also, blood serum was collected from each mouse. Both tumor TR:TREATMENT_SUMMARY and serum samples were stored at -80°C for further analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each collected serum sample (100 µL) was mixed with 300 µL of methanol SP:SAMPLEPREP_SUMMARY (≥99.9 %, LC-MS CHROMASOLV) in Eppendorf tubes, followed by vortex and SP:SAMPLEPREP_SUMMARY incubation at -20°C for 2 hours. After vortex and centrifugation at 14,000 rpm SP:SAMPLEPREP_SUMMARY for 15 minutes, the supernatant was evaporated at 35-40°C using speed vacuum SP:SAMPLEPREP_SUMMARY evaporation (EZ-2 Plus (GeneVac, Ipswich, UK). The extracted samples were SP:SAMPLEPREP_SUMMARY resuspended in 200 µL of 0.1% formic acid in Deionized Water-LC-MS CHROMASOLV. SP:SAMPLEPREP_SUMMARY Subsequently, the supernatant was filtered through a 0.45 µm pore size SP:SAMPLEPREP_SUMMARY hydrophilic nylon syringe filter for LC-MS/MS analysis and collected in inserts SP:SAMPLEPREP_SUMMARY within LC glass vials. All samples were placed in the autosampler at the SP:SAMPLEPREP_SUMMARY temperature set at 4℃ to proceed with the analysis by Q-TOF MS. A pooled QC SP:SAMPLEPREP_SUMMARY sample was created to ensure analysis reproducibility by mixing 10 µL from each SP:SAMPLEPREP_SUMMARY sample. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Mobile phases A (water with 0.1% formic acid) and B (acetonitrile with 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid) were used with the following gradient elusion mode: 0 to 2 min, 1% CH:CHROMATOGRAPHY_SUMMARY B; 2 to 17 min, 1–99% B; 17 to 20 min, 99% B; 20 to 20.1 min, 99–1% B; 20.1 CH:CHROMATOGRAPHY_SUMMARY to 30 min, 1% B. The flow rate was 0.25 mL/min from 0 to 20 min, 0.35 mL/min CH:CHROMATOGRAPHY_SUMMARY from 20 min to 28.3 min, and 0.25 mL/min from 28.3 to 30 min. The sample CH:CHROMATOGRAPHY_SUMMARY injection volume was 10 μl, and separation occurred on a Hamilton® Intensity CH:CHROMATOGRAPHY_SUMMARY Solo 2 C18 column (2.1 × 100 mm, 1.8 µm) (Bruker Daltonik) at 35°C. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Bruker Elute CH:COLUMN_NAME Hamilton Intensity Solo 2 C18 (100 × 2.1mm, 1.8um) CH:SOLVENT_A Water (0.1% Formic Acid) CH:SOLVENT_B ACN (0.1% Formic Acid) CH:FLOW_GRADIENT gradient elusion mode: 0 to 2 min, 1% B; 2 to 17 min, 1–99% B; 17 to 20 min, CH:FLOW_GRADIENT 99% B; 20 to 20.1 min, 99–1% B; 20.1 to 30 min, 1% B. CH:FLOW_RATE 0.25 mL/min from 0 to 20 min, 0.35 mL/min from 20 min to 28.3 min, and 0.25 CH:FLOW_RATE mL/min from 28.3 to 30 min. CH:COLUMN_TEMPERATURE 35 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker timsTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The ESI source conditions were set with a capillary voltage of 4500 V, drying MS:MS_COMMENTS gas flow rate of 10.0 l/min at 220°C, nebulizer pressure of 2.2 bar, and the MS:MS_COMMENTS End Plate offset at 500 V. Sodium formate (10 mM) was injected at the beginning MS:MS_COMMENTS of each sample run and used as a calibrant for internal calibration during data MS:MS_COMMENTS processing. The MS acquisition process consisted of two phases. First, an auto MS:MS_COMMENTS MS scan lasting from 0 to 0.3 minutes was utilized for calibrating sodium MS:MS_COMMENTS formate. The second phase encompassed auto MS/MS scanning with CID acquisition, MS:MS_COMMENTS including fragmentation, which extended from 0.3 to 30 minutes. Both acquisition MS:MS_COMMENTS phases were conducted in positive mode at a rate of 12 Hz. The automatic mass MS:MS_COMMENTS scan range within each run spanned from 50 to 1300 m/z, with a precursor ion MS:MS_COMMENTS width of ±0.5, a cycle time of 0.5 seconds, and a threshold of 400 counts. MS:MS_COMMENTS Active exclusion was initiated after three spectra and lifted after 0.2 minutes. MS:MS_COMMENTS For MS2 acquisition, a data-dependent acquisition (DDA) approach was employed, MS:MS_COMMENTS with collision energy settings varying between 100% and 250% and being set at 20 MS:MS_COMMENTS eV. TRX-2101/RT-28-calibrants from Nova Medical Testing Inc. for the Bruker MS:MS_COMMENTS T-ReX LC-QTOF were injected before sample analysis to assess the column's MS:MS_COMMENTS performance, reversed-phase liquid chromatography (RPLC) separation, multipoint MS:MS_COMMENTS retention time calibration, and the mass spectrometer. Additionally, MS:MS_COMMENTS TRX-3112-R/MS Certified Human serum solution for Bruker T-ReX LC-QTOF (provided MS:MS_COMMENTS by Nova Medical Testing Inc.) was prepared from pooled human blood and MS:MS_COMMENTS administered before sample analysis to validate the performance of the LC-MS MS:MS_COMMENTS instruments. The analysis followed a randomized sequence order, commencing with MS:MS_COMMENTS five injections of solvent A (0.1% formic acid in deionized water) to facilitate MS:MS_COMMENTS apparatus equilibration. Subsequently, five injections of the pooled QC sample MS:MS_COMMENTS were carried out. Furthermore, one QC injection was conducted every (9-10 MS:MS_COMMENTS samples) to assess the consistency of the analysis. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples 1A1-01-10368 1A1-02-10369 1A2-01-10370 1A2-02-10371 1A3-01-10372 1A3-02-10373 1B1-01-10374 1B1-02-10375 1B2-01-10376 1B2-02-10377 1B3-01-10378 1B3-02-10379 2A1-01-10381 2A1-02-10382 2A2-01-10383 2A2-02-10384 2B1-01-10385 2B1-02-10386 2B2-01-10387 2B2-02-10388 2B3-01-10389 2B3-02-10390 3A1-01-10392 3A1-02-10393 3A2-01-10394 3A2-02-10395 3B1-01-10396 3B1-02-10397 3B2-01-10398 3B2-02-10399 3B3-01-10400 3B3-02-10401 4A1-01-10403 4A1-02-10404 4A2-01-10405 4A2-02-10406 4A4-01-10407 4A4-02-10408 4B1-01-10409 4B1-02-10410 4B2-01-10411 4B2-02-10412 4B3-01-10413 4B3-02-10414 5A1-01-10416 5A1-02-10417 5A2-01-10418 5A2-02-10419 5A3-01-10420 5A3-02-10421 5B1-01-10422 5B1-02-10423 5B2-01-10424 5B2-02-10425 5B3-01-10426 5B3-02-10427 Factors Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:Doxorubicin Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Doxorubicin + 5-fluorouracil Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Positive Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control Treatment:Negative Control (R)-Lipoic acid 1702 1216 2030 1216 1118 870 1038 1058 1000 1356 1154 1160 1080 960 1400 1434 864 1014 1180 1110 1274 1162 1528 2002 1328 764 1348 1056 1070 1280 972 658 1426 1278 1008 1138 866 730 1070 1284 1258 1306 1226 1630 1062 1266 974 1342 962 854 1608 1240 1316 1222 1464 1090 1-Methyladenosine 1022 968 0 1260 1134 732 0 1084 1050 1642 0 1070 644 670 822 1068 1264 1244 796 1160 1022 1082 1166 776 980 884 940 744 1100 1146 1100 1038 1128 856 1238 1090 1246 1490 886 1032 1362 1320 1060 678 802 592 954 0 1144 886 1206 1020 1242 1260 946 884 1-Methylguanine 930 806 692 784 502 578 892 914 524 618 1338 956 720 858 926 830 496 504 1104 1134 1634 1426 228 290 196 284 322 276 262 252 488 268 854 696 896 978 476 786 702 832 834 974 440 700 772 830 1876 1810 984 688 974 974 1370 962 1306 1432 2,5-Furandicarboxylic acid 450 846 666 630 752 776 728 558 600 468 690 524 522 250 314 280 398 374 670 622 676 684 258 330 470 580 518 438 314 444 396 492 392 452 710 552 430 412 1274 974 712 642 626 912 474 520 846 1038 766 518 512 612 580 520 930 800 2-Hydroxy-3-methylbutyric acid 1118 1152 1002 804 646 892 874 0 1080 652 940 1062 1032 1162 1330 1320 868 916 1566 2262 1242 1208 1264 1362 894 962 1054 892 872 858 736 0 0 944 812 1264 902 632 874 1310 1014 1456 1074 1260 1016 958 1084 982 812 884 1048 946 1014 986 774 1118 3-Hexenedioic acid 7546 9444 8962 8838 10410 10498 9064 8806 8918 8808 10320 7126 6022 5914 6362 6534 8240 6202 7580 7360 10120 8188 6784 5846 7064 6984 7080 7810 6144 7086 9020 8858 9286 7096 10544 11334 10056 11210 11392 9700 8554 7870 9716 11812 7786 7668 9326 8568 8566 8202 9610 8180 8684 8654 10234 9556 3-Methylindole 5992 7310 6822 7018 6212 6108 6842 6128 5146 5030 7378 5930 3674 4072 4086 4974 5758 5240 5340 5688 5566 5338 6594 4734 5948 7064 5232 5728 5646 5070 5488 6054 5738 5830 6336 6276 4746 4454 6296 5758 6924 6818 7542 7522 5652 5326 7306 7292 6372 6256 5630 5972 5420 5702 5396 5882 5'-Methylthioadenosine 2960 3220 3132 2340 3230 3002 2968 2998 4072 4016 3072 2364 2466 1984 1846 2130 1838 1808 2248 2192 2480 2604 5244 4792 2294 1856 3074 3274 2002 2162 1712 2218 2544 2350 4404 4150 5328 4974 8062 6940 2616 3232 3462 3580 5064 4356 4628 4836 5494 4712 3046 3028 6480 4616 6714 6060 5-Hydroxy-L-tryptophan 668 814 742 830 886 924 658 766 806 1132 1126 824 216 308 726 658 696 622 688 596 608 484 1030 1110 794 644 668 798 864 816 874 932 720 926 910 1070 486 590 1480 1442 728 778 806 708 1294 1528 1830 1606 1732 2174 712 694 1382 1560 874 976 5-Hydroxytryptophol 692 642 672 672 578 836 518 638 838 576 1068 948 720 536 1054 1706 854 594 1692 1892 1110 840 800 774 832 886 886 938 868 856 1036 1258 496 1046 862 638 672 420 1512 1230 852 648 842 916 642 744 806 592 912 964 578 768 542 538 668 832 8,11,14-Eicosatrienoic acid 1072 1092 1168 1026 1048 958 1178 826 1014 1014 812 1138 950 1180 956 1184 1210 1456 866 852 1696 868 1116 1178 930 1066 1418 864 1226 1484 1018 1102 970 1418 1152 890 1092 1068 100 1064 952 1098 870 868 1682 982 946 892 1146 852 1118 876 1084 586 916 202 Acetone 2036 1958 1742 2196 1712 1426 1732 970 1928 2134 1620 1484 1102 1596 1518 2166 1844 2090 2002 2078 1444 1580 2718 1642 1500 2744 1568 1288 1354 1494 1356 1972 1358 2058 1478 1930 1236 1494 1378 1664 1266 1746 1304 1734 1222 2194 1332 1774 1184 2182 1542 1132 2322 1172 988 2212 Adenine 716 750 1646 2782 962 960 1008 750 640 948 782 436 784 498 1120 1262 490 502 1014 878 912 538 1022 976 628 628 702 800 788 974 1014 1112 476 524 1918 1812 1220 1090 3878 3194 1090 806 1402 1558 2564 2404 2140 2104 1726 1896 858 640 1228 998 1236 1028 Adenosine 3236 540 0 0 0 0 0 6206 8722 3416 0 3038 5338 4334 1408 1372 1866 2086 206 3088 0 0 398 13594 780 0 4836 0 2206 1968 2784 3224 8608 11962 17640 4694 6020 1866 174 16156 4238 4288 1610 0 10662 10566 8358 9974 22246 21064 6974 7408 10674 9436 12618 29754 Adenosine monophosphate 536 928 1142 698 872 1144 826 1038 2178 3378 740 860 1236 878 792 582 578 140 680 836 746 1080 35606 35872 456 408 1952 2084 446 600 602 582 2476 2322 8570 14364 1130 1324 2286 3512 1024 816 1022 928 3312 3932 2466 1962 9380 7656 1304 1192 2208 2784 8360 8340 Allantoic acid 1472 1308 1240 1384 2778 2428 1112 1012 728 750 1220 754 1644 1810 2816 3484 2102 2096 3068 3138 1398 1184 942 1108 1304 1122 1398 1334 2678 2874 2176 2900 968 1144 1542 1886 1380 1880 2434 2370 1276 1430 1366 1130 2796 2276 2974 2890 1774 2532 842 766 2126 2590 2440 1516 Androstenedione 0 1084 1072 1130 920 840 946 880 1034 826 710 1194 928 1018 798 1144 1112 0 860 834 944 702 878 736 666 774 738 966 1304 734 778 1070 896 0 0 910 702 880 698 984 0 692 742 766 0 758 746 900 1026 290 722 822 1100 770 0 924 Anserine 366 736 698 538 346 376 420 428 302 388 786 750 690 512 1154 876 394 350 1510 1130 1022 940 590 284 368 0 496 676 0 0 88 0 386 574 1044 1132 542 408 466 534 788 916 468 508 768 712 1646 1348 770 640 398 602 646 480 1158 1266 Aspartame 7138 6678 5520 5788 4340 4276 3990 4832 4196 3646 4700 5024 4850 4874 3682 4062 4784 4186 6276 7892 4178 4686 5714 4584 4374 3918 4526 4468 4928 5146 5196 4788 3524 3474 4342 4234 3848 3572 4968 4362 5192 6092 6182 7412 4354 4692 5018 4848 4370 3874 3694 3988 4638 4236 5056 4494 Asymmetric dimethylarginine 932 1040 820 1194 1110 980 1164 830 1280 1156 1332 1028 870 930 806 670 1016 792 536 556 880 776 482 804 776 816 546 512 872 910 970 994 1060 878 870 1272 1704 1562 666 744 1000 1050 948 1064 662 786 720 854 1006 798 1304 1048 980 948 714 1146 Benzoic acid 1252 2752 0 2716 3582 0 3850 0 0 3560 250 0 0 2234 2822 2810 0 1832 1708 3714 0 1702 330 2758 2886 1260 2538 0 0 0 314 1376 3326 294 3726 2322 1288 5230 290 1728 230 0 3560 0 412 0 314 1286 1922 0 0 0 0 3152 3766 340 Betaine 1912 1986 2368 2260 1738 1840 2336 1580 2390 2312 2046 1544 2944 3268 1742 2674 2986 3332 2554 2326 1312 1952 2024 1622 1908 1480 2134 2328 1422 1322 1532 1740 2130 2392 2526 2822 2622 2834 5462 4866 4062 3066 2652 3474 3964 3106 3450 4144 3116 2930 1942 2244 2566 2416 2610 3706 Biotin 710 1132 946 758 1546 976 918 1316 1194 966 1042 956 968 892 1068 1096 1020 1112 1176 1298 1206 1150 1232 938 884 788 1016 1296 1238 1044 990 1166 978 1136 872 996 1106 1118 1500 826 1160 1238 970 1048 1048 1006 862 984 1022 1150 994 842 942 824 1016 1128 Chlorpheniramine 522 710 992 590 574 702 658 624 500 568 734 546 864 676 0 494 894 1222 896 870 280 0 674 520 566 524 674 774 734 570 810 1028 616 568 1040 990 710 822 1026 884 740 1104 956 1012 766 860 1082 1058 1226 1002 566 596 624 558 700 778 Cortexolone 2074 2666 6910 4750 10630 10450 1050 1544 4176 5004 10828 8026 2184 2906 21666 17756 668 490 11862 12776 14464 14656 9738 8968 8634 9276 4880 5760 8906 8980 12502 10674 1914 2086 9848 11048 4308 3354 328 284 6210 6684 7226 8116 756 700 6316 6452 9842 12112 926 1110 0 8612 11704 11798 Cysteamine 336 946 886 968 784 1048 1216 530 590 1264 730 598 666 514 800 732 434 334 610 526 1180 1032 2314 1342 732 742 816 1188 412 594 564 384 586 564 748 1816 662 1040 2582 1556 672 562 1362 1298 880 1002 930 858 968 1040 570 520 610 716 948 1048 Cytidine 1424 2842 0 2094 2602 2250 1610 2530 2746 3738 2012 2194 2298 2552 1856 1618 1778 1390 1250 1468 2492 2756 2408 1596 2382 2232 2146 1490 1692 2002 2690 2966 3836 2264 3338 3002 2982 5066 1778 1656 1994 1684 2908 4188 2648 2292 4330 4996 2938 2976 2530 3140 2864 4404 3630 2458 Cytosine 2032 1888 2134 2174 2112 1914 1630 2102 1988 3108 1952 2066 1770 1950 1828 2586 2004 1826 2742 2142 2838 3200 1918 2290 2292 1958 1806 1976 1678 1794 2138 2236 2306 1934 1982 2484 3298 3874 2870 1806 1550 1662 2638 2744 2666 2242 3352 2490 2224 2700 2494 2126 2328 2998 2796 3084 D-Glucurono-6,3-lactone 412 1760 2310 1784 2466 1700 1226 0 302 0 1612 596 490 306 396 0 492 466 1268 258 2014 1676 3850 2150 0 1390 546 2806 238 486 248 402 520 564 584 18106 514 1580 5456 642 456 412 1940 2524 232 0 458 452 520 388 222 0 394 510 1620 400 Dehydroascorbic acid 734 868 504 618 292 538 634 768 838 774 228 688 500 0 332 272 706 830 360 382 562 228 0 0 520 706 430 244 734 508 896 634 676 684 584 508 614 640 200 680 874 590 618 314 610 590 806 680 818 1130 780 890 684 1012 652 674 Deoxycytidine 304 564 720 716 662 940 764 658 738 1142 1408 504 454 582 582 0 378 226 454 670 494 684 490 640 554 534 604 836 410 430 562 568 0 780 614 696 714 1474 666 588 512 440 680 578 468 0 798 862 860 512 498 416 380 894 834 448 Diaminopimelic acid 1038 550 676 624 982 698 796 1070 870 844 930 0 234 458 588 248 742 610 378 422 230 0 510 394 542 550 360 544 612 546 904 696 386 276 688 490 352 800 792 1026 502 410 782 650 370 486 390 430 748 548 822 894 640 700 434 568 DL-2-aminooctanoic acid 7780 8620 14986 16970 8768 8356 12194 11338 10482 11064 4466 3900 7018 5680 5998 6270 10204 10484 8264 8758 9454 9266 8736 6994 7792 7844 8176 7816 5448 5476 10706 11490 10782 10724 11426 12150 18746 18836 20778 17000 14840 14880 18678 20658 9390 8526 13418 15268 16364 10918 14652 14926 11784 11296 16846 14874 Epinephrine 3118 0 2860 2788 3320 3030 3182 4700 2554 2058 2760 2356 2196 2236 576 962 3476 3270 1778 1926 2928 2626 1586 1378 2386 2848 1878 2016 2788 1758 2622 2532 3862 3618 2984 2956 2194 2696 1506 1164 2470 2074 2362 2178 2090 1674 1520 2188 2006 2812 3088 2976 2932 2558 2628 1880 Ethanolamine 820 1438 1094 1104 886 640 1088 1044 944 918 1490 762 1198 1052 528 628 732 626 1152 848 1156 1166 680 640 850 660 1040 740 808 680 638 816 672 938 1010 654 1090 1148 1066 1196 1182 862 730 798 868 842 900 652 946 816 968 796 890 862 906 910 Fructose 6-phosphate 336 798 590 436 470 398 770 604 458 516 370 604 0 0 348 0 366 540 328 0 502 428 522 662 382 480 352 336 636 428 410 394 896 850 834 806 974 552 1792 1518 0 586 538 452 504 690 958 1062 338 444 762 620 550 652 786 574 Gluconolactone 0 0 0 0 0 0 0 474 818 368 0 556 338 256 0 0 0 416 0 0 0 0 0 196 0 0 298 0 486 496 594 564 0 600 582 0 1024 0 0 2044 882 536 0 0 1574 1298 1818 1750 0 1180 560 670 1530 0 298 1222 Glutathione 914 652 730 596 654 554 588 868 704 724 462 926 446 502 476 360 456 580 276 360 548 770 424 392 690 384 546 552 600 422 684 624 840 840 804 746 516 630 616 564 508 646 782 686 720 550 874 996 840 858 780 592 0 766 674 966 Glycerophosphocholine 13314 14666 15524 18700 16556 16446 17710 12376 9562 11048 11968 10410 14328 13154 9532 9724 10430 11868 11452 11616 10264 11462 7492 12752 15376 13722 14682 14084 15500 15362 13840 9596 11846 11058 9614 9650 8986 11418 8098 7266 10756 10760 14998 14942 13872 12676 16572 11558 10224 11230 11462 9680 8710 9508 11588 11558 Guanine 206 490 1646 402 324 2438 358 366 570 588 480 440 160 0 0 0 316 282 180 0 810 0 460 480 0 166 0 798 0 0 138 284 742 524 1656 1530 774 966 626 668 218 456 362 1326 494 512 244 406 1356 1562 364 444 810 1042 2804 1818 Guanosine 2208 2270 724 2412 1450 950 1550 3212 2678 3156 1282 3374 2072 1946 628 714 3616 3758 0 178 220 310 0 0 0 0 544 0 520 348 1786 2040 3842 4294 3822 3432 3836 4088 866 2026 3658 3344 1592 446 1678 2054 1820 1738 3428 3052 3168 2990 3302 3994 3088 3998 Guanosine monophosphate 214 870 790 796 982 810 1030 472 620 1268 1218 316 0 246 238 358 268 196 550 390 772 666 1220 824 388 422 234 1008 0 280 262 208 952 670 4086 4480 1620 1796 2002 1086 686 0 874 922 730 842 506 350 2428 2156 674 688 1212 1384 5208 3446 Homocitrulline 588 830 926 1042 914 542 0 450 692 336 676 716 452 442 294 0 476 562 0 198 290 470 312 514 788 896 572 0 634 866 534 566 802 886 882 534 0 394 588 540 852 766 1026 770 606 416 818 998 418 304 398 352 540 988 524 418 Hydroxyindoleacetic acid 700 732 1260 772 654 658 514 948 792 0 896 712 784 702 934 1064 438 724 0 1100 946 810 0 1196 874 966 1038 806 902 1036 0 886 572 756 724 592 1544 1622 1990 1642 1510 1290 658 670 0 0 1618 1284 1138 0 622 572 0 862 1162 980 Hypoxanthine 42480 35998 15698 37582 26662 15046 22436 39538 67672 69554 28206 48562 36282 33010 36140 35528 36750 35098 5168 6648 8358 18020 14262 37262 5350 2670 29460 368 16116 16602 32960 32544 41190 43220 54166 50904 33072 28786 39580 74950 53948 52594 32944 9620 36746 38882 48908 49366 55456 56052 43382 45604 49140 48338 54336 62786 Indole 37578 37300 42870 39126 34672 35458 33638 34086 32340 31888 46490 38822 21554 21198 23576 22520 28684 30122 28008 31504 30546 29326 32524 27974 34972 35936 28956 31772 32128 33414 28778 32052 34272 32910 37320 37954 25278 27980 34308 33048 36000 37540 38440 40634 33138 34504 41352 38584 39718 36530 27778 30566 31740 30702 31122 29080 Indole-3-carboxylic acid 994 842 1106 1094 808 968 968 794 1058 672 1130 676 1388 1658 1414 1498 1502 1334 1026 1272 688 748 636 654 860 1236 1138 1064 1014 998 978 916 898 840 1066 1016 1010 1048 528 460 1096 1754 1326 1154 652 750 728 504 472 614 1034 756 682 520 970 610 Indoleacrylic acid 211834 221948 244176 225558 197874 200350 191652 187154 179126 184962 256812 205694 122692 125456 138914 136164 170716 172960 165664 183050 180058 177078 172080 152708 198082 196780 153768 171496 185696 190268 170722 179830 202456 185238 206578 207754 154758 149430 199364 171062 204108 208342 218928 235700 186802 193974 230994 200716 220118 196142 164498 165248 171898 178476 169070 175424 Indolelactic acid 720 670 790 774 650 716 624 712 478 724 762 586 858 648 706 794 630 482 928 1066 1128 1362 810 600 614 770 696 1008 1340 966 480 582 460 526 598 0 772 504 608 534 548 718 496 470 638 460 502 578 0 598 610 602 662 728 470 568 Inosine 18018 12730 7220 17478 10852 4198 10446 17384 29956 24124 11108 21400 13832 12914 13264 12386 15482 14508 2086 2382 1600 5024 3164 11576 2234 1336 7936 450 7624 8336 12482 13522 19440 18028 22898 21098 13330 11306 10348 23848 20758 22298 14552 5328 14372 15444 20598 20388 23180 22662 17804 19466 21776 20306 23810 25960 Isovalerylcarnitine 12228 14466 17308 14926 13512 10220 9164 9680 7664 8750 12200 9624 10230 12422 11786 12830 6024 7016 12810 14472 14734 13794 8742 7514 8560 9144 10684 13172 14768 18008 10542 13616 6436 6966 14014 14376 11832 11740 22490 21250 17564 15672 11442 14062 9308 9486 14566 15512 8568 9658 9826 10264 10808 9200 14054 15032 Kynurenic acid 980 838 998 816 790 794 700 390 438 570 368 222 364 374 390 310 408 492 314 396 400 416 366 284 274 380 342 424 930 664 262 402 344 316 470 326 770 744 964 784 652 938 888 852 0 394 346 376 452 538 636 456 236 228 438 392 L-Acetylcarnitine 108178 136736 133376 124234 144500 153566 124858 136246 110842 132474 148428 120578 96030 94230 89456 92986 110044 92228 97230 102470 127784 129956 100206 96368 96890 98066 99204 112188 88552 93964 124616 129820 120170 112168 146544 149492 148472 161182 165836 137658 115504 114234 164026 180962 103784 103498 146180 126348 113166 114694 120506 118624 123296 126834 172946 143340 L-Arginine 5392 5838 6038 5380 770 1104 4536 4486 3466 4472 3766 4794 9592 8292 654 942 3786 3454 994 1150 7710 8942 7662 5730 4622 6772 7776 7510 1862 2290 652 538 4698 3182 1956 1434 3286 3430 0 0 4998 6060 3370 3634 0 0 0 0 0 362 3096 4320 318 380 760 1178 L-Carnitine 22476 17958 13402 18492 22436 15772 17378 21990 14602 13434 17350 14970 14164 14944 10008 10042 19012 18308 12150 13476 15718 19346 11408 11644 17428 18794 14044 19194 12250 13512 12134 15480 22482 28048 21708 19546 20814 19218 13414 13244 10200 10358 13688 16606 19924 18652 16440 15146 13162 13116 21084 16618 13840 15198 18784 14974 L-Fucose 1248 1226 1488 1050 972 1258 1690 1234 872 1054 1678 1220 1224 1136 1594 1736 846 688 2598 1672 3146 2508 586 504 438 322 366 618 0 0 0 198 1366 1400 1734 1844 988 1178 1270 1706 1738 1978 894 852 1244 1034 2620 2860 1158 1358 1842 2264 2396 1822 1972 1888 L-Kynurenine 602 292 700 974 464 622 674 470 782 842 664 590 784 714 0 1052 314 472 934 706 0 778 1068 554 826 0 980 578 814 906 666 290 854 748 390 464 1420 1116 1418 1282 1180 1374 558 672 876 798 1418 1494 0 1164 498 310 0 550 810 656 L-Leucine 6012 2600 172 2612 2730 392 1440 3782 4658 3690 3250 5378 5204 4906 5352 4334 4598 4726 3222 6936 0 2650 0 4090 2834 2210 4696 132 5524 6094 4862 4628 4754 4692 5522 3268 4224 2502 1046 5928 5978 6634 3642 1130 4276 4392 4884 5296 4708 4120 5136 4732 4084 3906 4834 3922 L-Methionine 2118 3554 3088 2760 2216 2212 2612 2348 2474 3414 3544 2408 1972 1826 1182 1318 2504 2158 1888 2134 2538 3024 1650 1786 1876 2372 2010 1396 1380 1344 1976 1784 1914 1696 2452 2086 2366 2456 1738 2404 2600 1888 2918 2022 1738 2240 2344 2006 2288 2100 1642 1920 2252 2864 2000 1578 L-Norleucine 1660 1708 1896 1734 1518 1706 1268 1340 1460 1782 1690 1164 974 1164 1040 1084 1824 1242 1560 1454 1300 870 1294 1112 1364 1120 1102 866 1558 1626 1274 1542 974 1372 1334 1988 1824 1408 1892 1996 2138 1940 1674 2284 1550 1306 2218 1664 1388 1708 1080 1136 1266 1490 1326 1434 L-Phenylalanine 18272 18592 19626 17538 14012 14346 12990 14608 12996 14824 16408 16026 22706 23682 26888 27752 16696 18258 30984 30836 24120 24286 23772 26038 19036 19658 22828 18020 20684 17408 16520 19342 17624 15392 18504 22122 13628 13056 20636 17960 20576 20936 18848 18168 15616 14266 18212 17716 12592 14968 19158 17846 16660 15378 17202 18644 L-Tryptophan 45754 45768 54308 46860 42218 42740 41604 40570 38938 41392 51040 40558 27516 26488 29042 27834 36534 36348 37412 38788 38944 36028 37306 33690 40744 40934 34964 38860 39270 40318 36672 36016 42534 40154 47072 44578 32310 33572 36834 37874 43934 43942 42338 50564 37824 42086 46016 44970 45328 43192 34502 35770 39558 36092 38442 36574 Levoglucosan 1852 1898 2246 2306 2078 2318 1992 1806 2072 2358 2246 1986 890 884 806 602 1400 1338 686 874 1332 1314 852 1108 1298 1326 858 1008 1942 1780 1960 1594 2742 2666 2432 2298 1470 1696 3020 3046 2424 1628 2236 2766 2032 2274 3260 3394 2126 1806 1676 1686 1892 2080 1980 1762 Levulinic acid 12006 11080 13668 12608 10732 11850 11060 10650 10198 10392 13744 11608 7066 7182 7418 6858 10434 9134 9564 9372 10222 9348 9626 8458 11248 10838 9038 9760 9936 10196 9736 10396 11368 10492 10000 11608 8744 7606 11104 10450 11726 12122 11744 12810 11736 11310 12034 12382 11776 12088 10434 9252 9624 10232 9944 9230 m-Coumaric acid 12196 1662 0 1196 272 0 230 9062 7716 3948 0 8752 3614 3342 3134 2980 3796 5268 1320 2638 0 0 0 2628 2366 0 2842 0 5906 5632 6356 5928 7582 8282 4256 3620 8088 3178 0 6958 11726 17340 2628 0 8044 7874 9474 9638 8334 8124 9450 10278 9062 8568 3136 8338 Methylmalonic acid 1266 0 1100 0 0 0 0 1724 2016 2150 0 1748 754 708 1150 0 896 0 0 0 0 0 2158 2648 218 0 1248 0 948 888 910 1240 1488 1354 2374 1848 1482 0 2586 2402 1726 1974 1612 0 1584 1224 1650 1838 1432 1606 1916 1546 1590 1348 1664 1968 N-Acetyl-L-phenylalanine 0 268 460 556 590 694 632 542 1368 1038 1480 1184 0 358 528 606 586 440 0 0 350 288 1098 1392 0 224 676 486 428 318 662 0 976 950 704 858 788 626 1736 1768 774 634 678 804 1006 962 676 644 1082 1082 878 1022 456 0 766 958 N-Methyl-a-aminoisobutyric acid 3508 3740 3502 3624 3806 2524 2572 2734 2548 3018 2880 2646 2112 2546 2766 3284 1990 2932 2896 3026 2634 2672 3060 3192 2710 2776 3910 2690 3264 3788 2634 3032 2256 2286 3610 3808 3330 2646 6128 6752 2784 3826 3426 3258 3298 3666 3828 4324 2924 2510 2884 2908 3410 3172 3486 2924 Naproxen 790 1270 820 1022 1026 980 1070 888 934 1014 1184 1288 1642 1276 1164 1048 1184 948 1228 1410 2064 1836 1174 1072 828 810 922 982 874 1018 996 1138 978 818 1024 1572 910 1096 0 0 940 1170 1172 1114 970 1112 1196 958 1532 920 470 644 1476 1512 1158 1036 Niacinamide 2118 1942 1778 1866 2304 2162 2176 2176 2962 2986 1380 1850 2390 2506 3358 3656 2592 1732 2236 2710 2456 3684 5756 7554 2112 1478 4294 2718 1676 1954 1844 2184 2818 2478 3626 3792 2834 2384 6972 6550 2836 2524 2828 0 5176 6094 5144 4648 3728 3678 2464 3106 3018 3898 2824 2966 o-Tyrosine 1592 1560 1286 1468 1120 1160 1086 984 928 1006 974 1146 522 466 1006 676 804 852 706 666 1128 1024 920 966 748 790 594 778 884 968 798 898 828 592 940 1240 1172 984 1136 920 1304 1766 1442 1326 838 804 1378 1210 1140 918 732 886 1132 978 948 1082 Ornithine 1626 1152 1350 1402 1304 1524 1322 1244 1042 1084 1148 774 690 602 848 932 508 570 920 602 670 586 422 624 602 542 680 554 678 500 404 346 558 352 412 316 410 508 1106 374 414 434 512 490 346 238 604 332 390 408 418 356 408 256 410 534 Pantothenic acid 12116 11260 11496 9326 11360 9878 8170 9950 8234 7418 10726 7788 6976 6494 8476 8486 6020 6422 10468 8462 14358 14722 7098 7966 6166 6436 6118 6710 5914 5578 6020 6668 7718 7094 10642 11452 8546 7786 9790 9428 8306 10724 9876 8738 6140 6474 9246 10038 8042 8226 10420 8266 9932 9078 10750 8582 Phenol 1424 0 0 0 0 0 0 1096 1428 0 0 0 826 0 530 0 908 692 0 492 0 0 0 248 274 0 0 0 586 620 816 896 1250 864 0 372 984 454 0 0 1424 1142 0 0 752 1010 1370 1556 864 698 1068 1168 966 1026 0 1216 Pyridine 1120 1604 2298 1490 1912 2164 1456 1346 1066 1416 1828 1484 1208 942 2306 2124 1116 806 1296 1530 3984 3520 7596 4686 984 1416 2006 2540 802 1006 978 1032 1266 1138 1792 4504 1132 1710 7920 4124 1162 1130 3306 3942 2218 2376 2530 3056 1598 2308 1200 1576 1482 1796 1912 1360 Pyrrolidonecarboxylic acid 796 714 1294 718 600 624 1244 464 750 826 750 926 658 524 788 602 640 1062 642 632 436 302 1008 1230 380 450 794 366 614 490 940 822 790 874 994 1114 1036 716 1264 1416 912 768 1006 1170 1394 1138 1226 1738 1054 1326 746 708 1176 1150 1198 1100 Saccharopine 2430 2098 2268 1908 2234 2154 1794 1706 1912 1818 1990 2326 1508 1460 1426 1416 1452 1494 1366 1644 1358 1276 1346 1246 1474 1542 1318 1306 1368 1564 1318 1386 1456 1228 1316 1462 1234 1128 1592 1626 1432 1054 1770 1368 1356 1348 1366 988 1490 1060 1458 1160 0 1200 1102 1100 Scopolamine 526 924 1054 768 800 698 726 486 616 1068 1084 556 456 184 100 0 694 268 536 430 0 592 590 464 526 602 350 366 566 618 308 572 360 368 588 742 704 1204 380 348 248 164 1034 958 504 428 474 350 536 454 454 492 392 778 606 302 Serotonin 3378 2650 1982 2868 2082 1516 1874 3148 2514 2304 1680 2908 1902 1908 864 844 2266 2746 550 604 376 494 188 428 1366 1334 308 162 2326 3668 2988 2956 2986 2946 2956 2536 2672 2902 1854 3292 3154 3232 4034 1720 2912 2346 3558 3264 2860 2512 3256 2922 3118 3144 3452 3332 Spermine 3986 4544 6020 4908 5162 5710 3890 4002 5342 3988 2896 1690 4938 4946 10952 12950 2086 2154 5586 5176 5952 5164 2126 2050 3104 2672 4044 3782 2446 2928 1924 2106 2204 1760 4414 4620 6824 7036 20508 17496 3528 3326 4140 4084 13664 12416 14256 12360 8312 8112 3768 4082 4366 5104 9700 6164 Sphinganine 12862 13702 16438 16076 5680 6106 15360 16952 18338 15812 21702 14088 25260 27024 11270 12858 33110 25870 36690 37292 76242 71644 3516 2632 2126 2238 3618 4652 1048 1224 970 1190 11194 10092 7502 8392 4334 3974 10604 7804 11892 12718 12164 14338 5944 4722 9586 8644 13644 11984 6454 7114 8696 9330 11764 12942 Sphingosine 6006 5726 7710 6344 5840 7244 5842 5680 5376 5448 4688 4080 6934 5660 5458 5524 4396 4596 3216 3080 8322 7640 20208 16540 2734 3308 4240 4084 6098 6186 2436 2640 4716 5022 6876 6682 18136 17538 12920 11446 11854 10120 11588 10944 7096 7294 8838 8106 4598 4782 4592 4250 3244 2524 6070 6274 Succinylacetone 2372 2068 1020 2122 1290 654 1084 1782 1220 1780 0 1492 1274 844 368 416 1250 1592 0 0 0 0 0 0 596 616 0 0 1452 1830 2292 2546 1826 2144 1658 1650 2212 2368 928 2124 2026 2130 2690 952 1456 1694 2376 2164 1436 1686 1970 2048 1796 1888 1546 1974 Taurine 422 556 760 494 1000 516 648 676 786 926 526 434 358 584 450 870 360 526 428 444 1212 1234 650 698 346 290 554 628 450 572 580 516 584 618 788 700 620 578 1270 1562 916 868 636 828 866 676 1052 890 798 974 806 596 970 848 828 1042 Taurocholic acid 0 0 0 0 224 314 0 0 184 172 0 0 0 0 0 0 1852 1870 0 0 0 0 0 0 276 360 0 480 724 0 64956 67874 0 846 52822 49272 0 858 5340 5644 222 362 342 434 1840 1422 32878 27862 37178 36464 198 302 0 0 75200 0 Trimethylamine 10036 13572 21360 12592 13964 15684 13124 14360 11474 15906 15710 11888 8646 7800 8408 8918 10258 9352 9006 9544 18302 12444 10176 10396 9220 9942 9236 10858 8544 9210 13066 11902 14090 10492 14310 15136 17206 16692 19756 14996 10326 12562 16304 18784 10198 10210 14734 15458 12766 12064 12754 12998 11936 12910 21332 15646 Tryptophanol 1044 990 1212 1222 906 120 1178 1348 540 510 934 1276 1006 1130 368 0 1540 1160 0 224 0 0 0 0 512 604 0 0 804 630 1198 1366 1098 1106 910 890 1492 1458 178 242 1150 984 1672 0 972 966 576 754 780 560 1302 1502 622 756 0 974 Urea 1912 2222 2268 2142 2596 2628 2136 2238 1426 2612 2172 1594 1584 1574 1468 1798 1784 1460 2048 2016 3054 2588 1816 2020 1876 2158 1750 1870 1806 1828 2666 1972 1602 1598 2468 2464 1714 1814 2486 2410 1946 1822 2262 2618 1726 1510 3608 1918 1660 1794 1852 2178 1164 1760 1812 2010 Uric acid 29182 30140 7236 0 22882 2344 0 20984 17310 16042 124 23756 17108 16930 23862 22070 17766 18500 35126 38052 0 20380 2466 10744 30744 0 18872 0 17494 17222 21058 20974 17664 16086 31088 22516 24044 24672 2280 11004 31718 33484 34636 0 10654 11202 12830 12508 11502 10654 23706 23076 15482 13622 27942 22992 Uridine 1234 1034 1198 0 1138 1166 1410 1344 1140 1300 1562 1054 1186 1196 1094 1752 1502 1212 1066 1536 1302 1372 1084 1452 1088 998 1012 1146 1114 934 940 1304 1116 1430 1084 1190 1172 914 1184 1002 1414 1214 1170 1132 1648 1014 0 1166 996 1248 1232 1320 1096 1184 1148 1446 Urocanic acid 850 748 1074 1020 950 604 594 550 922 1068 714 764 1330 1042 846 880 708 714 668 696 948 908 980 948 716 662 1150 1590 702 634 396 406 866 1052 610 760 1584 1852 744 730 1672 1338 608 748 568 750 964 910 604 674 1006 954 680 584 2008 1240 Xanthine 800 798 2486 2522 1480 1234 616 728 1236 988 1396 1254 328 518 628 656 474 396 914 660 588 704 1562 1558 534 700 774 510 636 630 772 758 1166 926 5160 5350 1012 1004 3302 4144 1586 1764 2804 2996 1524 1076 1912 1634 1216 1168 1474 1180 780 878 2216 2274 Xanthosine 574 998 2256 1860 1240 1228 546 532 866 908 1732 1142 302 350 550 548 276 198 920 846 658 586 1436 1662 666 712 728 424 752 746 918 806 1066 854 4926 4962 562 760 2694 3174 1504 1502 2288 1812 1040 938 2328 1506 716 1020 1970 1418 858 670 2010 2380 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Molecular Formula RT [min] m/z meas. M meas. MS/MS (R)-Lipoic acid C8H14O2S2 0.86 207.05075 206.04347 FALSE 1-Methyladenosine C11H15N5O4 2.1 282.11974 281.11247 TRUE 1-Methylguanine C6H7N5O 7.28 166.07758 165.07031 TRUE 2,5-Furandicarboxylic acid C6H4O5 1.49 157.01097 156.00369 TRUE 2-Hydroxy-3-methylbutyric acid C5H10O3 5.01 119.07248 118.0652 FALSE 3-Hexenedioic acid C6H8O4 1.7 145.04954 144.04227 TRUE 3-Methylindole C9H9N 5.89 132.08075 131.07347 TRUE 5'-Methylthioadenosine C11H15N5O3S 5.75 298.09687 297.0896 TRUE 5-Hydroxy-L-tryptophan C11H12N2O3 5.28 221.09212 220.08485 TRUE 5-Hydroxytryptophol C10H11NO2 6 178.08654 177.07927 TRUE 8,11,14-Eicosatrienoic acid C20H34O2 0.3 307.261 306.25372 FALSE Acetone C3H6O 0.31 59.04926 58.04199 TRUE Adenine C5H5N5 5.1 136.06166 135.05438 TRUE Adenosine C10H13N5O4 4.49 268.10397 267.09669 TRUE Adenosine monophosphate C10H14N5O7P 2 348.07022 347.06294 TRUE Allantoic acid C4H8N4O4 0.85 177.06115 176.05387 TRUE Androstenedione C19H26O2 20.92 287.19833 286.19105 FALSE Anserine C10H16N4O3 11.11 241.13359 240.12631 TRUE Aspartame C14H18N2O5 6.34 295.129 294.12172 TRUE Asymmetric dimethylarginine C8H18N4O2 1.33 203.15054 202.14327 FALSE Benzoic acid C7H6O2 2.45 123.04411 122.03684 TRUE Betaine C5H11NO2 1.16 118.0863 117.07903 TRUE Biotin C10H16N2O3S 0.3 245.09957 244.09229 FALSE Chlorpheniramine C16H19ClN2 12.23 275.12567 274.11839 TRUE Cortexolone C21H30O4 10.61 347.22196 346.21476 TRUE Cysteamine C2H7NS 2.09 78.0341 77.02682 FALSE Cytidine C9H13N3O5 1.59 244.09291 243.08564 TRUE Cytosine C4H5N3O 1.65 112.05041 111.04314 TRUE D-Glucurono-6,3-lactone C6H8O6 2.02 177.0333 176.02602 FALSE Dehydroascorbic acid C6H6O6 2.15 175.02348 174.01621 TRUE Deoxycytidine C9H13N3O4 2.04 228.09786 227.09059 TRUE Diaminopimelic acid C7H14N2O4 13.64 191.10671 190.09943 TRUE DL-2-aminooctanoic acid C8H17NO2 1.51 160.13333 159.12605 TRUE Epinephrine C9H13NO3 1.06 184.09458 183.0873 TRUE Ethanolamine C2H7NO 14.48 62.06018 61.0529 TRUE Fructose 6-phosphate C6H13O9P 1.33 261.03697 260.02969 TRUE Gluconolactone C6H10O6 3.33 179.04855 178.04128 TRUE Glutathione C10H17N3O6S 5.25 308.08591 307.07864 TRUE Glycerophosphocholine C8H20NO6P 0.94 280.0922 257.10304 TRUE Guanine C5H5N5O 2.09 152.05662 151.04935 TRUE Guanosine C10H13N5O5 4.7 284.09898 283.0917 TRUE Guanosine monophosphate C10H14N5O8P 2.07 364.06441 363.05714 TRUE Homocitrulline C7H15N3O3 2.07 190.11873 189.11146 FALSE Hydroxyindoleacetic acid C10H9NO3 4.94 192.06553 191.05826 TRUE Hypoxanthine C5H4N4O 4.7 137.04577 136.03845 TRUE Indole C8H7N 5.88 118.06497 117.05898 TRUE Indole-3-carboxylic acid C9H7NO2 1.46 162.05037 161.04309 FALSE Indoleacrylic acid C11H9NO2 5.87 188.07063 187.06338 TRUE Indolelactic acid C11H11NO3 8.22 206.08113 205.07385 TRUE Inosine C10H12N4O5 4.7 269.08805 268.08078 TRUE Isovalerylcarnitine C12H23NO4 6.24 246.17012 245.16285 TRUE Kynurenic acid C10H7NO3 6.47 190.05013 189.04285 TRUE L-Acetylcarnitine C9H17NO4 1.69 204.1231 203.11583 TRUE L-Arginine C6H14N4O2 1.11 175.11913 174.11186 TRUE L-Carnitine C7H15NO3 1.1 162.1126 161.10532 TRUE L-Fucose C6H12O5 7.28 165.06996 164.06269 TRUE L-Kynurenine C10H12N2O3 4.92 209.09224 208.08496 TRUE L-Leucine C6H13NO2 2.69 132.10186 131.09459 TRUE L-Methionine C5H11NO2S 2.08 150.0583 149.05102 TRUE L-Norleucine C6H13NO2 6.03 132.1019 131.09462 TRUE L-Phenylalanine C9H11NO2 5 166.08628 165.079 TRUE L-Tryptophan C11H12N2O2 5.86 205.09726 204.08998 TRUE Levoglucosan C6H10O5 1.27 163.06019 162.05291 TRUE Levulinic acid C5H8O3 5.88 117.05718 116.0499 TRUE m-Coumaric acid C9H8O3 2.86 165.05463 164.04737 TRUE Methylmalonic acid C4H6O4 2.28 119.03512 118.02784 TRUE N-Acetyl-L-phenylalanine C11H13NO3 5.38 208.09107 207.0838 TRUE N-Methyl-a-aminoisobutyric acid C5H11NO2 1.46 118.0862 117.07893 TRUE Naproxen C14H14O3 2.02 231.09788 230.0906 TRUE Niacinamide C6H6N2O 2.07 123.05524 122.04796 TRUE o-Tyrosine C9H11NO3 5.34 182.08095 181.07368 FALSE Ornithine C5H12N2O2 16.86 133.10123 132.09395 FALSE Pantothenic acid C9H17NO5 5.44 220.11803 219.11075 TRUE Phenol C6H6O 2.86 95.04907 94.04179 TRUE Pyridine C5H5N 2.07 80.04967 79.04239 TRUE Pyrrolidonecarboxylic acid C5H7NO3 4.92 130.04992 129.04264 TRUE Saccharopine C11H20N2O6 14.83 277.13512 276.12785 TRUE Scopolamine C17H21NO4 2.03 304.15037 303.14309 FALSE Serotonin C10H12N2O 4.7 177.10241 176.09513 TRUE Spermine C10H26N4 0.8 203.2232 202.21592 TRUE Sphinganine C18H39NO2 11.13 302.30558 301.2983 TRUE Sphingosine C18H37NO2 11.57 282.27934 299.28268 TRUE Succinylacetone C7H10O4 4.7 159.06791 158.06063 FALSE Taurine C2H7NO3S 1.26 126.02188 125.0146 TRUE Taurocholic acid C26H45NO7S 11.16 516.29969 515.29225 TRUE Trimethylamine C3H9N 1.7 60.0809 59.07362 TRUE Tryptophanol C10H11NO 4.9 162.09121 161.08394 TRUE Urea CH4N2O 1.2 61.03977 60.03249 TRUE Uric acid C5H4N4O3 2.12 169.03569 168.02841 TRUE Uridine C9H12N2O6 12.52 245.07858 244.0713 TRUE Urocanic acid C6H6N2O2 2 139.05015 138.04287 TRUE Xanthine C5H4N4O2 4.98 153.04073 152.03346 TRUE Xanthosine C10H12N4O6 5 285.08323 284.07595 TRUE METABOLITES_END #END