#METABOLOMICS WORKBENCH NReisdorphLab_20180226_162402 DATATRACK_ID:1318 STUDY_ID:ST001099 ANALYSIS_ID:AN001795 PROJECT_ID:PR000735 VERSION 1 CREATED_ON December 4, 2018, 10:09 am #PROJECT PR:PROJECT_TITLE Untargeted LC-MS to compare blood collection tube and processing time PR:PROJECT_SUMMARY Blood was collected from three healthy volunteers in 3 blood collection tubes: PR:PROJECT_SUMMARY serum separator tube SST (serum), EDTA (plasma), and P100 (plasma) and stored at PR:PROJECT_SUMMARY 4 degrees for 0, 0.5, 1, 2, 4, and 24 hours prior to centrifugation. Compounds PR:PROJECT_SUMMARY were extracted using liquid-liquid extraction to obtain a hydrophilic and a PR:PROJECT_SUMMARY hydrophobic fraction and analyzed using liquid chromatography mass spectrometry. PR:PROJECT_SUMMARY Differences among the blood collection tubes and sample processing time were PR:PROJECT_SUMMARY evaluated (ANOVA with Bonferroni FWER ≤ 0.05 and ANOVA with Benjamini Hochberg PR:PROJECT_SUMMARY FDR ≤ 0.1, respectively). PR:INSTITUTE University of Colorado, Denver PR:DEPARTMENT Pharmaceutical Sciences, Anschutz Medical Campus PR:LAST_NAME Reisdorph PR:FIRST_NAME Nichole PR:ADDRESS 12850 East Montview Blvd PR:EMAIL nichole.reisdorph@ucdenver.edu PR:PHONE 303-724-9234 #STUDY ST:STUDY_TITLE Untargeted LC-MS to compare blood collection tube and processing time – HILIC ST:STUDY_TITLE & C18 ST:STUDY_SUMMARY Blood was collected from three healthy volunteers in 3 blood collection tubes: ST:STUDY_SUMMARY serum separator tube SST (serum), EDTA (plasma), and P100 (plasma) and stored at ST:STUDY_SUMMARY 4 degrees for 0, 0.5, 1, 2, 4, and 24 hours prior to centrifugation. Compounds ST:STUDY_SUMMARY were extracted using liquid-liquid extraction to obtain a hydrophilic and a ST:STUDY_SUMMARY hydrophobic fraction and analyzed using liquid chromatography mass spectrometry. ST:STUDY_SUMMARY Differences among the blood collection tubes and sample processing time were ST:STUDY_SUMMARY evaluated (ANOVA with Bonferroni FWER ≤ 0.05 and ANOVA with Benjamini Hochberg ST:STUDY_SUMMARY FDR ≤ 0.1, respectively). ST:INSTITUTE University of Colorado, Denver ST:DEPARTMENT Anschutz Medical Campus ST:LAST_NAME Reisdorph ST:FIRST_NAME Nichole ST:ADDRESS 12850 East Montview Blvd ST:EMAIL nichole.reisdorph@ucdenver.edu ST:PHONE 3037249234 ST:NUM_GROUPS 17 ST:TOTAL_SUBJECTS 3 ST:NUM_MALES 2 ST:NUM_FEMALES 1 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 45-50 SU:GENDER Male and female SU:HUMAN_ETHNICITY Non-Hispanic white SU:HUMAN_SMOKING_STATUS Non-smoking #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_0min_EDTA Tube:EDTA | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_0min_P100 Tube:P100 | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_0min_Tiger Tube:Tiger | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_1-5hr_Tiger Tube:SST | Time:1.5 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_1hr_EDTA Tube:EDTA | Time:1 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_1hr_P100 Tube:P100 | Time:1 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_23-5hr_Tiger Tube:SST | Time:23.5 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_24hr_EDTA Tube:EDTA | Time:24 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_24hr_P100 Tube:P100 | Time:24 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_2hr_EDTA Tube:EDTA | Time:2 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_2hr_P100 Tube:P100 | Time:2 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_3-5hr_Tiger Tube:SST | Time:3.5 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_30min_EDTA Tube:EDTA | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_30min_P100 Tube:P100 | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_30min_Tiger Tube:SST | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_4hr_EDTA Tube:EDTA | Time:4 SUBJECT_SAMPLE_FACTORS Sbj1 sbj1_4hr_P100 Tube:P100 | Time:4 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_0min_EDTA Tube:EDTA | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_0min_P100 Tube:P100 | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_0min_Tiger Tube:Tiger | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_1-5hr_Tiger Tube:SST | Time:1.5 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_1hr_EDTA Tube:EDTA | Time:1 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_1hr_P100 Tube:P100 | Time:1 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_23-5hr_Tiger Tube:SST | Time:23.5 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_24hr_EDTA Tube:EDTA | Time:24 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_24hr_P100 Tube:P100 | Time:24 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_2hr_EDTA Tube:EDTA | Time:2 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_2hr_P100 Tube:P100 | Time:2 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_3-5hr_Tiger Tube:SST | Time:3.5 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_30min_EDTA Tube:EDTA | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_30min_P100 Tube:P100 | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_30min_Tiger Tube:SST | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_4hr_EDTA Tube:EDTA | Time:4 SUBJECT_SAMPLE_FACTORS Sbj2 sbj2_4hr_P100 Tube:P100 | Time:4 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_0min_EDTA Tube:EDTA | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_0min_P100 Tube:P100 | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_0min_Tiger Tube:Tiger | Time:Control_0 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_1-5hr_Tiger Tube:SST | Time:1.5 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_1hr_EDTA Tube:EDTA | Time:1 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_1hr_P100 Tube:P100 | Time:1 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_23-5hr_Tiger Tube:SST | Time:23.5 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_24hr_EDTA Tube:EDTA | Time:24 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_24hr_P100 Tube:P100 | Time:24 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_2hr_EDTA Tube:EDTA | Time:2 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_2hr_P100 Tube:P100 | Time:2 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_3-5hr_Tiger Tube:SST | Time:3.5 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_30min_EDTA Tube:EDTA | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_30min_P100 Tube:P100 | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_30min_Tiger Tube:SST | Time:0.5 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_4hr_EDTA Tube:EDTA | Time:4 SUBJECT_SAMPLE_FACTORS Sbj3 sbj3_4hr_P100 Tube:P100 | Time:4 #COLLECTION CO:COLLECTION_SUMMARY Blood from 3 healthy volunteers (40-50 years old) was collected in 3 types of CO:COLLECTION_SUMMARY vacutainers: Tiger vacutainer containing clot activator and gel to obtain serum, CO:COLLECTION_SUMMARY ethylenediaminetetraacetic acid (EDTA) vacutainer to obtain plasma, and P100(TM) CO:COLLECTION_SUMMARY vacutainer containing EDTA with protein stabilizers to obtain plasma. The Tiger CO:COLLECTION_SUMMARY top serum separator tubes required 30 minutes at room temperature prior to CO:COLLECTION_SUMMARY centrifugation to allow clot formation. Blood samples in the plasma tubes were CO:COLLECTION_SUMMARY left on ice for 0, 0.5, 1, 2, 4, or 24 hours. Blood samples in the serum tubes CO:COLLECTION_SUMMARY were left on ice for 0, 0.5, 1.5, 3.5, or 23.5 hours. Samples were then CO:COLLECTION_SUMMARY centrifuged at 10,000 x g at 4°C. The resulting serum or plasma was aliquoted CO:COLLECTION_SUMMARY into pre-chilled microcentrifuge tubes and stored at -80°C. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS Described in summary #TREATMENT TR:TREATMENT_SUMMARY N/A #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma and serum samples were thawed and briefly vortexed. 100μL of each sample SP:SAMPLEPREP_SUMMARY were transferred to a 1.5 mL microcentrifuge tube and kept at 0°C. 10μL of SP:SAMPLEPREP_SUMMARY hydrophobic and hydrophilic standards and spikes at room temperature were added SP:SAMPLEPREP_SUMMARY and the samples vortexed for 10 seconds. 400μL of ice-cold methanol was added. SP:SAMPLEPREP_SUMMARY Tubes were vortexed for 10 seconds and then centrifuged for 15 minutes at 0°C SP:SAMPLEPREP_SUMMARY at 18,000 rpm. Supernatant was transferred to a clean glass culture tube using a SP:SAMPLEPREP_SUMMARY plastic pipette. Samples were dried in glass culture tubes placed under N2 at SP:SAMPLEPREP_SUMMARY 35°C for approximately 1 hour. Using a glass pipette, 3 mL of methyl tert-butyl SP:SAMPLEPREP_SUMMARY ether (MTBE) was added to dried methanol residue in each glass culture tube. SP:SAMPLEPREP_SUMMARY Tubes were vortexed for 30 seconds. 750 μL of water was added, and tubes were SP:SAMPLEPREP_SUMMARY vortexed for 10 seconds. Tubes were centrifuged for 10 minutes at room SP:SAMPLEPREP_SUMMARY temperature at 1,000 rpm. 2.5 mL of the resulting MTBE layer (hydrophobic SP:SAMPLEPREP_SUMMARY fraction) was transferred to a new glass culture tube; the remaining layer was SP:SAMPLEPREP_SUMMARY the hydrophilic fraction. 3.0 mL of MTBE was added to the remaining hydrophilic SP:SAMPLEPREP_SUMMARY fraction, and tubes were vortexed for 10 seconds. These tubes were again SP:SAMPLEPREP_SUMMARY centrifuged for 10 minutes at room temperature at 1,000 rpm. 3.0 mL of MTBE was SP:SAMPLEPREP_SUMMARY aspirated and combined with the first MTBE layer. The MTBE fractions were dried SP:SAMPLEPREP_SUMMARY under N2 at 35°C and immediately re-suspended in 200 μL of methanol. Each tube SP:SAMPLEPREP_SUMMARY was vortexed for 5 seconds, transferred to a glass auto-sampler vial with a SP:SAMPLEPREP_SUMMARY glass insert using a Pasteur pipette. Samples were stored at -80°C. The SP:SAMPLEPREP_SUMMARY hydrophilic fractions were dried under N2 at 35°C. 100 μL of water and 400 μL SP:SAMPLEPREP_SUMMARY of ice-cold methanol were added to the dried hydrophilic fraction. Tubes were SP:SAMPLEPREP_SUMMARY vortexed for 10 seconds and spun immediately at medium speed for 1 minute. SP:SAMPLEPREP_SUMMARY Supernatants from each tube were transferred to a 1.5 mL microcentrifuge tube SP:SAMPLEPREP_SUMMARY using a Pasteur pipette. These tubes were then stored at -80°C for 25 minutes SP:SAMPLEPREP_SUMMARY then spun for 15 minutes at 0°C and 18,000 rpm. The supernatant was transferred SP:SAMPLEPREP_SUMMARY to a new 1.5 mL microcentrifuge tube using a plastic pipette. These tubes were SP:SAMPLEPREP_SUMMARY dried in a speed vacuum at 45°C and re-suspended in 100 μL of 95:5 water: SP:SAMPLEPREP_SUMMARY acetonitrile. Each tube was vortexed for 30 seconds and transferred to a glass SP:SAMPLEPREP_SUMMARY auto-sampler vial with a glass insert using a plastic pipette tip. Samples were SP:SAMPLEPREP_SUMMARY stored at -80°C. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 6520 CH:COLUMN_NAME Phenomenex Luna NH2 (250 x 2.0mm, 5um) CH:FLOW_RATE 0.5ml/min CH:COLUMN_TEMPERATURE 20 CH:SOLVENT_A 50% ACN with 20 mM ammonium acetate pH 9.45 CH:SOLVENT_B 100% ACN CH:SAMPLE_INJECTION 2uL CH:CAPILLARY_VOLTAGE 4000 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_VOLTAGE 4000 V MS:DRY_GAS_FLOW 10.0 L/min MS:DRY_GAS_TEMP 300 C MS:NEBULIZER 25 psi MS:MS_RESULTS_FILE ST001099_AN001795_Results.txt UNITS:Intensity (Log2 scale) Has m/z:Yes Has RT:Yes RT units:Minutes #END