#METABOLOMICS WORKBENCH FJR_20241019_012821 DATATRACK_ID:5295 STUDY_ID:ST003652 ANALYSIS_ID:AN005998 PROJECT_ID:PR002262 VERSION 1 CREATED_ON January 7, 2025, 5:13 am #PROJECT PR:PROJECT_TITLE Multiplatform Metabolomic Profiling of the Unilateral Ureteral Obstruction PR:PROJECT_TITLE Murine Model of CKD PR:PROJECT_SUMMARY In chronic kidney disease (CKD) research, animal models provide invaluable PR:PROJECT_SUMMARY insights into the disease’s etiopathogenesis and progression, particularly PR:PROJECT_SUMMARY through the evaluation of renal tissue. The unilateral ureteral obstruction PR:PROJECT_SUMMARY (UUO) rodent model stands out for its widespread use in CKD studies, due to its PR:PROJECT_SUMMARY advantages to generate renal fibrosis and accelerated mimicry of obstructive PR:PROJECT_SUMMARY nephropathy in humans. Despite its extensive use, the molecular underpinnings PR:PROJECT_SUMMARY driving kidney disease progression remain incompletely understood. Given the PR:PROJECT_SUMMARY crucial interplay between metabolism and fibrosis in CKD, a thorough examination PR:PROJECT_SUMMARY of the UUO renal tissue through metabolomics is required. Untargeted PR:PROJECT_SUMMARY multiplatform analysis enables a comprehensive measurement of the sample PR:PROJECT_SUMMARY metabolic profile, ensuring a maximum coverage of metabolite diversity to yield PR:PROJECT_SUMMARY extensive insights into the metabolism of this renal injury model. Therefore, in PR:PROJECT_SUMMARY this study, murine kidney tissue from the UUO model underwent analysis using PR:PROJECT_SUMMARY three separation techniques—liquid chromatography (LC), gas chromatography PR:PROJECT_SUMMARY (GC), and capillary electrophoresis (CE)—coupled with mass spectrometry (MS). PR:PROJECT_SUMMARY The findings reveal metabolic changes associated with tubulointerstitial PR:PROJECT_SUMMARY fibrosis, impacting essential pathways such as the TCA cycle, urea cycle, PR:PROJECT_SUMMARY polyamine metabolism, amino acids, one-carbon metabolism, purine catabolism, and PR:PROJECT_SUMMARY NAD+ synthesis, among others. Furthermore, fibrosis significantly influences the PR:PROJECT_SUMMARY renal tissue's lipidomic profile, characterized by a general decrease in most PR:PROJECT_SUMMARY lipid classes and an increase in glycerophospholipids with ether substituents, PR:PROJECT_SUMMARY hexosylceramides, and cholesterol esters compared to the control. These results PR:PROJECT_SUMMARY underscore the relevance of the untargeted multiplatform approach to obtain a PR:PROJECT_SUMMARY comprehensive overview of the alterations within the renal metabolic map, paving PR:PROJECT_SUMMARY the way for further exploration of the molecular mechanisms underlying CKD. PR:INSTITUTE Universidad CEU San Pablo PR:LAST_NAME Rupérez PR:FIRST_NAME Francisco PR:ADDRESS Campus Montepríncipe. Ctra. M-501 km 0 PR:EMAIL ruperez@ceu.es PR:PHONE 913724753 #STUDY ST:STUDY_TITLE Multiplatform Metabolomic Profiling of the Unilateral Ureteral Obstruction ST:STUDY_TITLE Murine Model of CKD ST:STUDY_SUMMARY In chronic kidney disease (CKD) research, animal models provide invaluable ST:STUDY_SUMMARY insights into the disease’s etiopathogenesis and progression, particularly ST:STUDY_SUMMARY through the evaluation of renal tissue. The unilateral ureteral obstruction ST:STUDY_SUMMARY (UUO) rodent model stands out for its widespread use in CKD studies, due to its ST:STUDY_SUMMARY advantages to generate renal fibrosis and accelerated mimicry of obstructive ST:STUDY_SUMMARY nephropathy in humans. Despite its extensive use, the molecular underpinnings ST:STUDY_SUMMARY driving kidney disease progression remain incompletely understood. Given the ST:STUDY_SUMMARY crucial interplay between metabolism and fibrosis in CKD, a thorough examination ST:STUDY_SUMMARY of the UUO renal tissue through metabolomics is required. Untargeted ST:STUDY_SUMMARY multiplatform analysis enables a comprehensive measurement of the sample ST:STUDY_SUMMARY metabolic profile, ensuring a maximum coverage of metabolite diversity to yield ST:STUDY_SUMMARY extensive insights into the metabolism of this renal injury model. Therefore, in ST:STUDY_SUMMARY this study, murine kidney tissue from the UUO model underwent analysis using ST:STUDY_SUMMARY three separation techniques—liquid chromatography (LC), gas chromatography ST:STUDY_SUMMARY (GC), and capillary electrophoresis (CE)—coupled with mass spectrometry (MS). ST:STUDY_SUMMARY The findings reveal metabolic changes associated with tubulointerstitial ST:STUDY_SUMMARY fibrosis, impacting essential pathways such as the TCA cycle, urea cycle, ST:STUDY_SUMMARY polyamine metabolism, amino acids, one-carbon metabolism, purine catabolism, and ST:STUDY_SUMMARY NAD+ synthesis, among others. Furthermore, fibrosis significantly influences the ST:STUDY_SUMMARY renal tissue's lipidomic profile, characterized by a general decrease in most ST:STUDY_SUMMARY lipid classes and an increase in glycerophospholipids with ether substituents, ST:STUDY_SUMMARY hexosylceramides, and cholesterol esters compared to the control. These results ST:STUDY_SUMMARY underscore the relevance of the untargeted multiplatform approach to obtain a ST:STUDY_SUMMARY comprehensive overview of the alterations within the renal metabolic map, paving ST:STUDY_SUMMARY the way for further exploration of the molecular mechanisms underlying CKD. ST:INSTITUTE Universidad CEU San Pablo ST:LAST_NAME Rupérez ST:FIRST_NAME Francisco ST:ADDRESS Campus Montepríncipe. Ctra. M-501 km 0 ST:EMAIL ruperez@ceu.es ST:PHONE 913724753 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 1WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=1WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=1WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=2WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=2WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=3WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=3WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=4WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=4WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=5WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=5WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=6WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=6WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=12WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=12WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-CT_CEMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=13WT-CT_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-UUO_CEMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=13WT-UUO_CEMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=1WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=1WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=2WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=2WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=3WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=3WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=4WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=4WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=5WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=5WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=6WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=6WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=12WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=12WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-CT_LCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=13WT-CT_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 13WT-UUO_LCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=13WT-UUO_LCMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=1WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 1WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=1WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=2WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 2WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=2WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=3WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 3WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=3WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=4WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 4WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=4WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=5WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 5WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=5WT-UUO_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 6WT-CT_GCMS Sample source:Kidney | Factor:CT RAW_FILE_NAME(Raw file name)=6WT-CT_GCMS.mzXML SUBJECT_SAMPLE_FACTORS - 12WT-UUO_GCMS Sample source:Kidney | Factor:UUO RAW_FILE_NAME(Raw file name)=12WT-UUO_GCMS.mzXML #COLLECTION CO:COLLECTION_SUMMARY All animal-related procedures and sample collections were conducted at the CO:COLLECTION_SUMMARY Centro de Biología Molecular "Severo Ochoa" (CBMSO) in accordance with the CO:COLLECTION_SUMMARY guidelines outlined in Directive 2010/63/EU of the European Parliament, known as CO:COLLECTION_SUMMARY the Guide for the Care and Use of Laboratory Animals. The study (PROEX 098.0/22) CO:COLLECTION_SUMMARY received approval from the CBMSO Ethics Committee for Animal Experimentation, CO:COLLECTION_SUMMARY the ethics committee of the Consejo Superior de Investigaciones Científicas CO:COLLECTION_SUMMARY (CSIC) and the Regulatory Unit for Animal Experimental Procedures of the CO:COLLECTION_SUMMARY Comunidad de Madrid. Mice were housed in a specific pathogen-free animal CO:COLLECTION_SUMMARY facility at CBMSO, adhering to EU regulations. CO:SAMPLE_TYPE Kidney #TREATMENT TR:TREATMENT_SUMMARY The UUO murine model (UUO7d) was stablished using the surgical procedure TR:TREATMENT_SUMMARY described in previous studies. Briefly, mice were anesthetized with isoflurane TR:TREATMENT_SUMMARY and the left ureter was doubly ligated with subsequent cutting between the TR:TREATMENT_SUMMARY ligatures. Adequate analgesia was provided to these animals after surgery and TR:TREATMENT_SUMMARY sacrifice was carried out after seven days using CO2 overdose. Control and TR:TREATMENT_SUMMARY obstructed kidneys were harvested following perfusion with PBS. Kidney samples TR:TREATMENT_SUMMARY were categorized into two experimental groups: wild-type control (WTCT) and TR:TREATMENT_SUMMARY wild-type obstruction (WTOBS), each containing 8 samples. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY All analytical-grade organic solvents and chemicals utilized in this study were SP:SAMPLEPREP_SUMMARY obtained from Merck/Sigma-Aldrich (Germany), VWR International/BHD Prolabo SP:SAMPLEPREP_SUMMARY (Spain) and Agilent Technologies (USA). Sialylation-grade pyridine was procured SP:SAMPLEPREP_SUMMARY from VWR International BHD Prolabo (Spain). Reference mass solutions for LC-MS SP:SAMPLEPREP_SUMMARY and CE-MS were obtained from Agilent Technologies. Deionized water (Milli-Q) SP:SAMPLEPREP_SUMMARY utilized throughout the study was obtained using a Milli-Q PLUS system from SP:SAMPLEPREP_SUMMARY Millipore (Austria). Sample treatment Each kidney was dissected both lengthwise SP:SAMPLEPREP_SUMMARY and crosswise to obtain four equal pieces. These sections were promptly frozen SP:SAMPLEPREP_SUMMARY in liquid nitrogen and stored at -80ºC until further analysis. Sample treatment SP:SAMPLEPREP_SUMMARY For tissue disruption and homogenization, a cold methanol solution of 50% was SP:SAMPLEPREP_SUMMARY added to achieve a tissue-weight volume ratio of 1:10. Homogenization was SP:SAMPLEPREP_SUMMARY conducted using a TissueLyser LT bead-mill homogenizer device (QIAGEN, Hilden, SP:SAMPLEPREP_SUMMARY Germany) with 2.8 mm (mean diameter) steel beads 1. The homogenization process SP:SAMPLEPREP_SUMMARY entailed vibrating at a maximum power of 50Hz for 5 minutes, carried out over 4 SP:SAMPLEPREP_SUMMARY cycles, with a 1-minute interval on ice between each cycle. The weight range of SP:SAMPLEPREP_SUMMARY the kidney tissue varied between 25 and 50 mg (WTCT, 32–52.8 mg; WTOBS, SP:SAMPLEPREP_SUMMARY 27.4–50.5 mg). Once the homogenate was obtained, the metabolite extraction SP:SAMPLEPREP_SUMMARY process followed different protocols depending on the analytical platform used SP:SAMPLEPREP_SUMMARY 2,3. For CE-MS analysis, 100 µL of 0.2M formic acid was added to 100 µL of SP:SAMPLEPREP_SUMMARY homogenate, followed by vortex-mixing and centrifugation (16,000×g for 10 SP:SAMPLEPREP_SUMMARY minutes at 4 °C). The resulting supernatant was then transferred to a SP:SAMPLEPREP_SUMMARY Centrifree ultracentrifugation device (Milipore Ireland Ltd., Cork, Ireland) SP:SAMPLEPREP_SUMMARY equipped with a 30-kDa protein cutoff filter for deproteinization through SP:SAMPLEPREP_SUMMARY centrifugation (2000×g for 70 minutes at 4 °C). The filtrate obtained was SP:SAMPLEPREP_SUMMARY transferred to a chromacol vial and subsequently evaporated using a SpeedVac SP:SAMPLEPREP_SUMMARY Concentrator (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the residue SP:SAMPLEPREP_SUMMARY was resuspended in 50 μL of 0.1 M formic acid containing 0.2 mM methionine SP:SAMPLEPREP_SUMMARY sulfone as an internal standard (IS). In the case of LC-MS and GC-MS analysis, SP:SAMPLEPREP_SUMMARY 320 μL of cold methanol was added to 100 μL of homogenate, followed by the SP:SAMPLEPREP_SUMMARY addition of 80 μL of methyl tert-butyl ether. The mixture was vortex-mixed for SP:SAMPLEPREP_SUMMARY 1 hour at room temperature. Subsequently, the samples were centrifuged at 4000 g SP:SAMPLEPREP_SUMMARY at 20ºC for 20 minutes, and the resulting supernatants were divided for LC-MS SP:SAMPLEPREP_SUMMARY and GC-MS analysis. For LC-MS analysis, 100 μL of the supernatant was directly SP:SAMPLEPREP_SUMMARY injected into the UHPLC-MS system. Regarding GC-MS analysis, 300 μL of the SP:SAMPLEPREP_SUMMARY supernatant was transferred to a chromacol vial and evaporated to dryness using SP:SAMPLEPREP_SUMMARY a SpeedVac Concentrator system. The derivatization process commenced by SP:SAMPLEPREP_SUMMARY reconstituting the samples with 20 μL of O-methoxyamine hydrochloride (15 SP:SAMPLEPREP_SUMMARY ng/mL) in pyridine for the methoximation step. After vigorous ultrasonication SP:SAMPLEPREP_SUMMARY and vortex-mixing, the vials were incubated in the dark at room temperature for SP:SAMPLEPREP_SUMMARY 16 hours. Subsequently, for the silylation step, 20 μL of BSTFA:TMCS (99:1) was SP:SAMPLEPREP_SUMMARY added, followed by vortex-mixing for 5 minutes and incubation at 70ºC in the SP:SAMPLEPREP_SUMMARY oven for 1 hour. Finally, 100 μL of heptane containing 20 ppm of tricosane (IS) SP:SAMPLEPREP_SUMMARY was added. It is worth mentioning that due to the limited availability of SP:SAMPLEPREP_SUMMARY samples, the GC-MS analysis was performed with only 6 samples in each group. SP:SAMPLEPREP_SUMMARY Quality control samples (QCs) were prepared for each analytical technique by SP:SAMPLEPREP_SUMMARY pooling equal volumes of each homogenized kidney tissue. Blank samples, which SP:SAMPLEPREP_SUMMARY lacked the analyte of interest but underwent the same extraction protocols, were SP:SAMPLEPREP_SUMMARY also prepared alongside the experimental samples. These blank samples were SP:SAMPLEPREP_SUMMARY analyzed at the beginning and at end of the analytical sequence. QC samples were SP:SAMPLEPREP_SUMMARY injected at the beginning of the sequence to condition and stabilize the SP:SAMPLEPREP_SUMMARY analysis system, and subsequently after every 5-6 experimental samples SP:SAMPLEPREP_SUMMARY throughout the run to assess the stability and performance of the analysis, and SP:SAMPLEPREP_SUMMARY finally, at the end of the analysis sequence. All experimental samples were SP:SAMPLEPREP_SUMMARY randomized prior to sample preparation and analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The GC-MS analysis was carried out utilizing an Agilent 7890B GC instrument CH:CHROMATOGRAPHY_SUMMARY coupled with a 7250 QTOF mass spectrometer system (Agilent Technologies). The CH:CHROMATOGRAPHY_SUMMARY analysis conditions were consistent with those described earlier . In summary, 1 CH:CHROMATOGRAPHY_SUMMARY μL of the derivatized sample was injected through an Agilent DB5-MS GC CH:CHROMATOGRAPHY_SUMMARY Capillary Column (30 m length, 0.25 mm, 0.25 µm film 95% CH:CHROMATOGRAPHY_SUMMARY dimethylpolysiloxane/5% diphenylpolysiloxane) using an Agilent autosampler CH:CHROMATOGRAPHY_SUMMARY (7693A). The samples were injected in a split ratio of 1:10 into a Restek 20782 CH:CHROMATOGRAPHY_SUMMARY deactivated glass-wool split liner. The injector port was established at 250ºC, CH:CHROMATOGRAPHY_SUMMARY the flow rate of helium carrier gas was set at 1 mL/min through the column. The CH:CHROMATOGRAPHY_SUMMARY temperature gradient was programmed at 60ºC as initial oven temperature CH:CHROMATOGRAPHY_SUMMARY (maintained for 1 min), increasing to 325ºC at a rate of 10ºC per minute. This CH:CHROMATOGRAPHY_SUMMARY temperature was kept for 10 min before cooling down. The total time of the CH:CHROMATOGRAPHY_SUMMARY analysis run was 37.5 minutes. CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890A CH:COLUMN_NAME Agilent DB5-MS (30m x 0.25mm, 0.25um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE 1 mL/min CH:COLUMN_TEMPERATURE Programmed Temperature Gradient #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7250 MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The parameters used in the Agilent 7250 QTOF mass spectrometer system were: an MS:MS_COMMENTS electron ionization (EI) source set at 70 eV EI energy, 200ºC in the filament MS:MS_COMMENTS source temperature, 280ºC in the detector transfer line temperature, and 150ºC MS:MS_COMMENTS as quadrupole temperature. Finally, the mass spectrometer collected mass spectra MS:MS_COMMENTS data within a 40-600 m/z range at a scan rate of 10 spectra/s MS:MS_RESULTS_FILE ST003652_AN005998_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END