#METABOLOMICS WORKBENCH tcavallo_20161229_082410 DATATRACK_ID:805 STUDY_ID:ST000545 ANALYSIS_ID:AN000831 PROJECT_ID:PR000399 VERSION 1 CREATED_ON January 31, 2017, 11:49 am #PROJECT PR:PROJECT_TITLE CHEAR Plasma Reference Material Proficiency Test Biocrates PR:PROJECT_TYPE CHEAR-UT-1 PR:PROJECT_SUMMARY CHEAR Reference Material Plasma was provided by Emory University. The material PR:PROJECT_SUMMARY was prepared and analyzed by way of LC-MS and Biocrates workflow employed by the PR:PROJECT_SUMMARY Eastern Regional Metabolomics Resource Core (protocols available in metabolomics PR:PROJECT_SUMMARY workbench). Six samples were injected of the sample reference material prepared PR:PROJECT_SUMMARY in replicate. PR:INSTITUTE RTI International PR:DEPARTMENT Discovery-Sciences-Technology (DST) PR:LABORATORY RTI CHEAR Analytical Hub - Untargeted Analysis Resource Core (UARC) 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 Plasma Reference Material Proficiency Test Biocrates ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY CHEAR Reference Material Plasma was provided by Emory University. The material ST:STUDY_SUMMARY was prepared and analyzed by way of LC-MS and Biocrates workflow employed by the ST:STUDY_SUMMARY Eastern Regional Metabolomics Resource Core (protocols available in metabolomics ST:STUDY_SUMMARY workbench). Six samples were injected of the sample reference material prepared ST:STUDY_SUMMARY 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 27709 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 - PP_A_09_1 Sample Type:Replicate CHEAR ID=CHE000054; Aliquot ID=1001; Matrix=Plasma; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_94_0_1_1_00_1010986041.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_94_1_1_1_00_1010986041.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_94_0_1_1_00_1010986041.wiff SUBJECT_SAMPLE_FACTORS - PP_A_09_2 Sample Type:Replicate CHEAR ID=CHE000054; Aliquot ID=1002; Matrix=Plasma; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_11_0_1_1_00_1010986055.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_11_1_1_1_00_1010986055.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_11_0_1_1_00_1010986055.wiff SUBJECT_SAMPLE_FACTORS - PP_A_09_3 Sample Type:Replicate CHEAR ID=CHE000054; Aliquot ID=1003; Matrix=Plasma; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_23_0_1_1_00_1010986060.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_23_1_1_1_00_1010986060.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_23_0_1_1_00_1010986060.wiff SUBJECT_SAMPLE_FACTORS - PP_A_09_4 Sample Type:Replicate CHEAR ID=CHE000054; Aliquot ID=1004; Matrix=Plasma; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_35_0_1_1_00_1010986074.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_35_1_1_1_00_1010986074.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_35_0_1_1_00_1010986074.wiff SUBJECT_SAMPLE_FACTORS - PP_A_09_5 Sample Type:Replicate CHEAR ID=CHE000054; Aliquot ID=1005; Matrix=Plasma; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_47_0_1_1_00_1010986089.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_47_1_1_1_00_1010986089.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_47_0_1_1_00_1010986089.wiff SUBJECT_SAMPLE_FACTORS - PP_A_09_6 Sample Type:Replicate CHEAR ID=CHE000054; Aliquot ID=1006; Matrix=Plasma; Biocrates AbsoluteIDQ® p180 KIT2 (LCMS) filename=KIT2-0-5404_1010986211_59_0_1_1_00_1010986093.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- POS) filename=KIT3-0-5404_1010986230_59_1_1_1_00_1010986093.wiff; Biocrates AbsoluteIDQ® p180 KIT3 (FIA- NEG) filename=KIT3-0-5404_1010986230_59_0_1_1_00_1010986093.wiff SUBJECT_SAMPLE_FACTORS - PBS_01 Sample Type:Control CHEAR ID=PBS; 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=PBS; 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=PBS; 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=Standards; 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=Standards; 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=Standards; 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=Standards; 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=Standards; 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=Standards; 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=Standards; 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=Standards; 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=Standards; 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=Standards; 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 Plasma was provided by Emory University. #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Parent Aliquots were quickly prepared in the 4°C room using the Drummond pipet SP:SAMPLEPREP_SUMMARY aid and serological pipet. 40 mL plasma were transferred to 50 mL tubes in 4°C SP:SAMPLEPREP_SUMMARY room and capped immediately and placed on ice. If needed, the bulk plasma was SP:SAMPLEPREP_SUMMARY mixed in between aliquots. The parent aliquots were labeled appropriately. SP:SAMPLEPREP_SUMMARY Sub-Aliquots were prepared on ice at the bench. The parent aliquots were mixed SP:SAMPLEPREP_SUMMARY by inverting the tube thoroughly before and in between aliquots as needed. 1 mL SP:SAMPLEPREP_SUMMARY plasma was transferred to cryovials and capped immediately and stored on ice SP:SAMPLEPREP_SUMMARY until sample splitting was competed. The sub aliquots were labeled SP:SAMPLEPREP_SUMMARY appropriately. Sub-aliquots were stored at -80°C. Plasma Aliquots for LCMS SP:SAMPLEPREP_SUMMARY platforms: Used sub-aliquot “PP_A_09” to prepare aliquots for various LCMS SP:SAMPLEPREP_SUMMARY platforms. Allowed sub-aliquot to thaw on ice for 30 – 60 min. Vortexed SP:SAMPLEPREP_SUMMARY aliquot briefly on vortexer, centrifuged at 4 °C for 2 minutes at 16,000 rcf. SP:SAMPLEPREP_SUMMARY Aliquoted out 6 tubes for Biocrates from “PP_A_09”. Aliquots were stored at SP:SAMPLEPREP_SUMMARY -80 °C until analysis. Sample preparation for Biocrates Plate: CHEAR plasma 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. Biocrates Plate Preparation: A Biocrates p180 kit was prepared SP:SAMPLEPREP_SUMMARY following the AbsoluteIDQ™ p180 Kit metabolomics procedure. Briefly, an SP:SAMPLEPREP_SUMMARY internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC SP:SAMPLEPREP_SUMMARY standards and calibration standards were added to their corresponding wells. The SP:SAMPLEPREP_SUMMARY samples (20 µL) were then added to the appropriate wells and dried for 30 SP:SAMPLEPREP_SUMMARY minutes under nitrogen flow. The plate was derivatized using a 5% SP:SAMPLEPREP_SUMMARY phenylisothiocyanate (PITC) solution in (1:1:1) ethanol:pyridine:water and, SP:SAMPLEPREP_SUMMARY then, incubated for 20 minutes followed by a drying step under nitrogen flow. An SP:SAMPLEPREP_SUMMARY extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. SP:SAMPLEPREP_SUMMARY The plate was then shaken and centrifuged. After centrifugation, 150 µL was SP:SAMPLEPREP_SUMMARY removed and transferred to a second 96-well plate (LCMS plate). This second SP:SAMPLEPREP_SUMMARY plate was diluted with 150 µL of HPLC grade water for a subsequent LCMS (MRM SP:SAMPLEPREP_SUMMARY analysis) for measuring amino acids and biogenic amines. All wells in the SP:SAMPLEPREP_SUMMARY original plate were diluted with 400 µL of flow injection analysis (FIA) SP:SAMPLEPREP_SUMMARY Running Solvent for a FIA-MS (MRM analysis) for measuring lipids, SP:SAMPLEPREP_SUMMARY acylcarnitines, and hexose. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE FIA CH:INSTRUMENT_NAME Agilent 1100 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_SPRAY_VOLTAGE -4500 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS um MS_METABOLITE_DATA_START Samples p180 QC1 PP_A_09_5 p180 Cal1 PP_A_09_3 p180 Cal7 p180 QC2 PP_A_09_4 PP_A_09_6 PBS_01 p180 Cal4 PP_A_09_2 p180 Cal5 PBS_02 p180 Cal2 PBS_03 p180 QC3 p180 Cal3 p180 Cal6 Factors Sample Type:Control Sample Type:Replicate Sample Type:Control Sample Type:Replicate Sample Type:Control 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:Control Sample Type:Control Sample Type:Control Sample Type:Control H1 5277 3531 5833 624 6012 800 10958 5363 5709 3.29 1046 5680 884 6.6 785 4.29 17444 1159 813 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name H1 METABOLITES_END #END