#METABOLOMICS WORKBENCH tcavallo_20161229_123004 DATATRACK_ID:806 STUDY_ID:ST000544 ANALYSIS_ID:AN000828 PROJECT_ID:PR000398 VERSION 1 CREATED_ON January 30, 2017, 6:06 pm #PROJECT PR:PROJECT_TITLE CHEAR Urine Reference Material Proficiency Testing Biocrates PR:PROJECT_TYPE CHEAR-UT-1 PR:PROJECT_SUMMARY CHEAR Reference Material Urine. The material was prepared and analyzed by way PR:PROJECT_SUMMARY of LC-MS and Biocrates workflow employed by the Eastern Regional Metabolomics PR:PROJECT_SUMMARY Resource Core (protocols available in metabolomics workbench). Six samples were PR:PROJECT_SUMMARY injected of the sample reference material prepared in replicate. PR:INSTITUTE RTI International PR:DEPARTMENT Discovery-Sciences-Technology (DST) PR:LABORATORY RTI CHEAR Analytical Hub - Untargeted Analysis Resource Core PR:LAST_NAME Fennell PR:FIRST_NAME Timothy PR:ADDRESS 3040 E Cornwallis Road, Research Triangle Park, NC 27709 PR:EMAIL fennell@rti.org PR:PHONE 919-485-2781 #STUDY ST:STUDY_TITLE CHEAR Urine Reference Material Proficiency Test Biocrates ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY CHEAR Reference Material Urine. The material was prepared and analyzed by way ST:STUDY_SUMMARY of LC-MS and Biocrates workflow employed by the Eastern Regional Metabolomics ST:STUDY_SUMMARY Resource Core (protocols available in metabolomics workbench). Six samples were ST:STUDY_SUMMARY injected of the sample reference material prepared in replicate. ST:INSTITUTE RTI International ST:DEPARTMENT Discovery-Sciences-Technology (DST) ST:LABORATORY RTI CHEAR Analytical Hub - Untargeted Analysis Resource Core ST:LAST_NAME Fennell ST:FIRST_NAME Timothy ST:ADDRESS 3040 E Cornwallis Road, Research Triangle Park, NC ST:EMAIL fennell@rti.org ST:PHONE 919-485-2781 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - UP_A_03_1 Sample Type:Replicate CHEAR ID=CHE000066; Aliquot ID=401; Matrix=Urine; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_71_0_1_1_00_1010986104.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_71_1_1_1_00_1010986104.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_71_0_1_1_00_1010986104.wiff SUBJECT_SAMPLE_FACTORS - UP_A_03_2 Sample Type:Replicate CHEAR ID=CHE000066; Aliquot ID=402; Matrix=Urine; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_83_0_1_1_00_1010986119.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_83_1_1_1_00_1010986119.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_83_0_1_1_00_1010986119.wiff SUBJECT_SAMPLE_FACTORS - UP_A_03_3 Sample Type:Replicate CHEAR ID=CHE000066; Aliquot ID=403; Matrix=Urine; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_95_0_1_1_00_1010986123.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_95_1_1_1_00_1010986123.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_95_0_1_1_00_1010986123.wiff SUBJECT_SAMPLE_FACTORS - UP_A_03_4 Sample Type:Replicate CHEAR ID=CHE000066; Aliquot ID=404; Matrix=Urine; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_12_0_1_1_00_1010986138.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_12_1_1_1_00_1010986138.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_12_0_1_1_00_1010986138.wiff SUBJECT_SAMPLE_FACTORS - UP_A_03_5 Sample Type:Replicate CHEAR ID=CHE000066; Aliquot ID=405; Matrix=Urine; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_24_0_1_1_00_1010986142.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_24_1_1_1_00_1010986142.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_24_0_1_1_00_1010986142.wiff SUBJECT_SAMPLE_FACTORS - UP_A_03_6 Sample Type:Replicate CHEAR ID=CHE000066; Aliquot ID=406; Matrix=Urine; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_36_0_1_1_00_1010986157.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_36_1_1_1_00_1010986157.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_36_0_1_1_00_1010986157.wiff SUBJECT_SAMPLE_FACTORS - PBS_01 Sample Type:Control CHEAR ID=1; Aliquot ID=1; Matrix=PBS; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_13_0_1_1_10_11000002.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_13_1_1_1_10_11000002.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_13_0_1_1_10_11000002.wiff SUBJECT_SAMPLE_FACTORS - PBS_02 Sample Type:Control CHEAR ID=2; Aliquot ID=2; Matrix=PBS; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_25_0_1_1_10_11000002.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_25_1_1_1_10_11000002.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_25_0_1_1_10_11000002.wiff SUBJECT_SAMPLE_FACTORS - PBS_03 Sample Type:Control CHEAR ID=3; Aliquot ID=3; Matrix=PBS; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_37_0_1_1_10_11000002.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_37_1_1_1_10_11000002.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_37_0_1_1_10_11000002.wiff SUBJECT_SAMPLE_FACTORS - p180 Cal1 Sample Type:Control CHEAR ID=1; Aliquot ID=1; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_49_0_1_1_02_20000611.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_49_1_1_1_02_20000611.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_49_0_1_1_02_20000611.wiff SUBJECT_SAMPLE_FACTORS - p180 Cal2 Sample Type:Control CHEAR ID=2; Aliquot ID=2; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_61_0_1_1_13_20000612.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_61_1_1_1_13_20000612.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_61_0_1_1_13_20000612.wiff SUBJECT_SAMPLE_FACTORS - p180 Cal3 Sample Type:Control CHEAR ID=3; Aliquot ID=3; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_73_0_1_1_14_20000613.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_73_1_1_1_14_20000613.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_73_0_1_1_14_20000613.wiff SUBJECT_SAMPLE_FACTORS - p180 Cal4 Sample Type:Control CHEAR ID=4; Aliquot ID=4; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_85_0_1_1_15_20000614.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_85_1_1_1_15_20000614.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_85_0_1_1_15_20000614.wiff SUBJECT_SAMPLE_FACTORS - p180 Cal5 Sample Type:Control CHEAR ID=5; Aliquot ID=5; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_02_0_1_1_16_20000615.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_02_1_1_1_16_20000615.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_02_0_1_1_16_20000615.wiff SUBJECT_SAMPLE_FACTORS - p180 Cal6 Sample Type:Control CHEAR ID=6; Aliquot ID=6; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_14_0_1_1_17_20000616.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_14_1_1_1_17_20000616.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_14_0_1_1_17_20000616.wiff SUBJECT_SAMPLE_FACTORS - p180 Cal7 Sample Type:Control CHEAR ID=7; Aliquot ID=7; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_26_0_1_1_18_20000617.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_26_1_1_1_18_20000617.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_26_0_1_1_18_20000617.wiff SUBJECT_SAMPLE_FACTORS - p180 QC1 Sample Type:Control CHEAR ID=1; Aliquot ID=1; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_38_0_1_1_03_721029.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_38_1_1_1_03_721029.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_38_0_1_1_03_721029.wiff SUBJECT_SAMPLE_FACTORS - p180 QC2 Sample Type:Control CHEAR ID=2; Aliquot ID=2; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_50_0_1_1_04_721046.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_50_1_1_1_04_721046.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_50_0_1_1_04_721046.wiff SUBJECT_SAMPLE_FACTORS - p180 QC3 Sample Type:Control CHEAR ID=3; Aliquot ID=3; Matrix=Standards; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_62_0_1_1_05_721050.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_62_1_1_1_05_721050.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_62_0_1_1_05_721050.wiff #COLLECTION CO:COLLECTION_SUMMARY CHEAR Reference Material Urine #TREATMENT TR:TREATMENT_SUMMARY No treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Urine: Parent and Sub-Aliquots Removed bulk urine from -80°C storage and thawed SP:SAMPLEPREP_SUMMARY urine by storing it at 4°C overnight. The following day, the thawed urine was SP:SAMPLEPREP_SUMMARY inspected to ensure there was no ice remaining and the bulk urine was then mixed SP:SAMPLEPREP_SUMMARY in the 4°C room by repeatedly inverting the container. Parent Aliquots were SP:SAMPLEPREP_SUMMARY quickly prepared in the 4°C room using the Drummond pipet aid and serological SP:SAMPLEPREP_SUMMARY pipet. 42 mL urine were transferred to 50 mL tubes in 4°C room and capped SP:SAMPLEPREP_SUMMARY immediately and placed on ice. If needed, the bulk urine was mixed in between SP:SAMPLEPREP_SUMMARY aliquots. The parent aliquots were labeled appropriately. Sub-Aliquots were SP:SAMPLEPREP_SUMMARY prepared on ice at the bench. The parent aliquots were mixed by inverting the SP:SAMPLEPREP_SUMMARY tube thoroughly before and in between aliquots as needed. 2.5 mL urine was SP:SAMPLEPREP_SUMMARY transferred to 5 mL cryovials and capped immediately and stored on ice until SP:SAMPLEPREP_SUMMARY sample splitting was competed. The sub aliquots were labeled appropriately. SP:SAMPLEPREP_SUMMARY Sub-aliquots were stored at -80°C. Urine Aliquots for LCMS platforms: Used SP:SAMPLEPREP_SUMMARY sub-aliquot “UP_A_03” to prepare aliquots for various LCMS platforms. SP:SAMPLEPREP_SUMMARY Allowed sub-aliquot to thaw on ice for 30 – 60 min. Vortexed aliquot briefly SP:SAMPLEPREP_SUMMARY on vortexer, centrifuged at 4 °C for 2 minutes at 16,000 rcf. Aliquoted out 6 SP:SAMPLEPREP_SUMMARY tubes for Biocrates (30 µL each) from “UP_A_03”. Aliquots were stored at SP:SAMPLEPREP_SUMMARY -80 °C until analysis. Sample preparation for Biocrates Plate: CHEAR urine SP:SAMPLEPREP_SUMMARY samples were thawed on ice for 30–60 min. All samples were vortexed on a SP:SAMPLEPREP_SUMMARY multi-tube vortexer for 4 mins at 5,000 rpm and centrifuged at 4 °C for 5 SP:SAMPLEPREP_SUMMARY minutes at 16,000 rcf before loading on the p180 plate. The samples were then SP:SAMPLEPREP_SUMMARY placed on ice in the analysis order for sample loading on the p180 Biocrates SP:SAMPLEPREP_SUMMARY plate. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE FIA CH:INSTRUMENT_NAME AB Sciex 4000 QTRAP LC/MS/MS CH:COLUMN_NAME No Column CH:SOLVENT_B Methanol with Biocrates FIA solvent CH:COLUMN_PRESSURE 4-50 bar CH:SAMPLE_INJECTION 20uL CH:ANALYTICAL_TIME 3.0 minutes CH:TARGET_SAMPLE_TEMPERATURE 10 degrees C CH:SAMPLE_LOOP_SIZE 100uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 4000 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:COLLISION_GAS Medium MS:ION_SOURCE_TEMPERATURE 500 degrees C MS:ION_SPRAY_VOLTAGE -4500 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS uM MS_METABOLITE_DATA_START Samples p180 Cal1 UP_A_03_5 UP_A_03_4 p180 Cal7 p180 QC2 UP_A_03_2 PBS_01 p180 Cal4 p180 Cal5 UP_A_03_6 PBS_02 p180 Cal2 PBS_03 p180 QC3 UP_A_03_1 UP_A_03_3 p180 Cal3 p180 Cal6 Factors Sample Type:Control Sample Type:Replicate Sample Type:Replicate Sample Type:Control Sample Type:Control Sample Type:Replicate Sample Type:Control Sample Type:Control Sample Type:Control Sample Type:Replicate Sample Type:Control Sample Type:Control Sample Type:Control Sample Type:Control Sample Type:Replicate Sample Type:Replicate Sample Type:Control Sample Type:Control H1 3531 624 7539 7697 800 10958 8001 3.29 1046 884 8366 6.6 785 4.29 17444 7263 8006 1159 813 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID H1 METABOLITES_END #END