Summary of Study ST000610

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000446. The data can be accessed directly via it's Project DOI: 10.21228/M8DC72 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST000610
Study Title	CHEAR Urine Reference Material
Study Type	Broad spectrum, reverse phase LCMS metabolomics
Study Summary	CHEAR UrineRef_20160721 was provided by Emory University. The material was prepared and analyzed using the Metabolomics Reverse Phase Broad Spectrum analysis on a Synapt G2-Si system. Six samples were injected of the sample reference material that were prepared in replicate.
Institute	RTI International
Department	ACP
Last Name	Fennell
First Name	Timothy
Address	3040 E Cornwallis Rd, Durham, North Carolina, 27709, USA
Email	fennell@rti.org
Phone	9194852781
Submit Date	2017-05-16
Num Groups	1
Total Subjects	6
Raw Data Available	Yes
Raw Data File Type(s)	raw(Waters)
Chear Study	Yes
Analysis Type Detail	LC-MS
Release Date	2024-12-31
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR000446
Project DOI:	doi: 10.21228/M8DC72
Project Title:	CHEAR Plasma Reference Material
Project Type:	Broad spectrum, reverse phase LCMS metabolomics
Project Summary:	CHEAR PlasmaRef_20160726 was provided by Emory University. The material was prepared and analyzed using the Metabolomics Reverse Phase Broad Spectrum analysis on a Synapt G2-Si system. Six samples were injected of the sample reference material that were prepared in replicate.
Institute:	RTI International
Department:	APC
Last Name:	Fennell
First Name:	Timothy
Address:	3040 E Cornwallis Rd, Durham, North Carolina, 27709, USA
Email:	fennell@rti.org
Phone:	9194852781
Funding Source:	U2CES026544

Subject:

Subject ID:	SU000633
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Subject Comments:	Urine
Species Group:	Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Matrix
SA033592	UP_A_03_12	Urine
SA033593	UP_A_03_11	Urine
SA033594	UP_A_03_10	Urine
SA033595	UP_A_03_8	Urine
SA033596	UP_A_03_9	Urine
SA033597	UP_A_03_7	Urine

Showing results 1 to 6 of 6

Showing results 1 to 6 of 6

Collection:

Collection ID:	CO000627
Collection Summary:	N/A
Sample Type:	Urine
Storage Conditions:	-80˚C

Treatment:

Treatment ID:	TR000647
Treatment Summary:	N/A

Sample Preparation:

Sampleprep ID:	SP000640
Sampleprep Summary:	Urine: Parent and Sub-Aliquots The CHEAR UrineRef_20160721 (500 mL) was removed from -80°C storage and thawed by storing it at 4°C overnight. The following day, the thawed urine was inspected to ensure there was no ice remaining and the bulk urine was then mixed in the 4°C room by repeatedly inverting the container. Parent aliquots were quickly prepared in the 4°C room using the Drummond pipet aid and serological pipet. 42 mL urine aliquots were transferred to 50 mL tubes in 4°C room and capped immediately and placed on ice. If needed, the bulk urine was mixed in between aliquots. The parent aliquots were labeled appropriately. Sub-aliquots were prepared on ice at the bench. The parent aliquots were mixed by inverting the tube thoroughly before and in between aliquots as needed. 2.5 mL urine aliquots were transferred to 5 mL cryovials and capped immediately and stored on ice until sample splitting was competed. The sub aliquots were labeled appropriately. Sub-aliquots were stored at -80°C. Urine Aliquots for LCMS platforms: The sub-aliquot UP_A_03 was used to prepare aliquots for various LCMS platforms. UP_A_03 was thawed on ice for 30 – 60 min and vortexed aliquot briefly on a vortexer, followed by centrifugation at 4 °C for 2 minutes at 16,000 rcf. Six aliquots of 60 µL volumes were prepared for Reverse Phase analysis from UP_A_03. Aliquots were stored at -80 °C until analysis. Sample preparation for Reverse Phase: The 6 urine aliquots were thawed on ice for 30–60 min, and then 50 µL of each sample was transferred into a new pre-labeled Lo-Bind Eppendorf tube. A volume of 400 µL methanol containing 0.025 mg/mL tryptophan-d5 as the internal standard was added to extract metabolites by vortex for 2 mins at 5000 rpm. Sample solution was centrifuged at 16,000 rcf for 4 min at room temperature to precipitate proteins. A volume of 350 µL of supernatant was transferred to pre-labeled 2.0 mL Lo-bind tubes and dried down using a SpeedVac system (LabConco CentriVap) at room temperature. A volume of 100 µL of Water/Methanol solution (95:5) was added to reconstitute each sample. Samples were then thoroughly mixed by vortex for 10 mins at 5000 rpm and then centrifuged at 16,000 rcf for 4 min at room temperature. The supernatants were transferred to pre-labeled autosampler vials before analysis. LC-MS analysis: The prepared urine samples were analyzed by an LC-MS system comprised of a Waters Acquity UPLC and a Synapt G2Si ESI-Q-TOF mass spectrometer. Chromatographic separation was accomplished on an Acquity HSST3 C18 column (2.1 X 100 mm, 1.8 µm) at 50 °C using a gradient elution comprised by mobile phase A: 0.1% formic acid-water (v/v), and mobile phase B: 0.1% formic acid-methanol (v/v), using a flow rate of 0.400 mL/min. The injection volume was 5 µL.

Sampleprep ID:

SP000640

Sampleprep Summary:

Urine: Parent and Sub-Aliquots The CHEAR UrineRef_20160721 (500 mL) was removed from -80°C storage and thawed by storing it at 4°C overnight. The following day, the thawed urine was inspected to ensure there was no ice remaining and the bulk urine was then mixed in the 4°C room by repeatedly inverting the container. Parent aliquots were quickly prepared in the 4°C room using the Drummond pipet aid and serological pipet. 42 mL urine aliquots were transferred to 50 mL tubes in 4°C room and capped immediately and placed on ice. If needed, the bulk urine was mixed in between aliquots. The parent aliquots were labeled appropriately. Sub-aliquots were prepared on ice at the bench. The parent aliquots were mixed by inverting the tube thoroughly before and in between aliquots as needed. 2.5 mL urine aliquots were transferred to 5 mL cryovials and capped immediately and stored on ice until sample splitting was competed. The sub aliquots were labeled appropriately. Sub-aliquots were stored at -80°C. Urine Aliquots for LCMS platforms: The sub-aliquot UP_A_03 was used to prepare aliquots for various LCMS platforms. UP_A_03 was thawed on ice for 30 – 60 min and vortexed aliquot briefly on a vortexer, followed by centrifugation at 4 °C for 2 minutes at 16,000 rcf. Six aliquots of 60 µL volumes were prepared for Reverse Phase analysis from UP_A_03. Aliquots were stored at -80 °C until analysis. Sample preparation for Reverse Phase: The 6 urine aliquots were thawed on ice for 30–60 min, and then 50 µL of each sample was transferred into a new pre-labeled Lo-Bind Eppendorf tube. A volume of 400 µL methanol containing 0.025 mg/mL tryptophan-d5 as the internal standard was added to extract metabolites by vortex for 2 mins at 5000 rpm. Sample solution was centrifuged at 16,000 rcf for 4 min at room temperature to precipitate proteins. A volume of 350 µL of supernatant was transferred to pre-labeled 2.0 mL Lo-bind tubes and dried down using a SpeedVac system (LabConco CentriVap) at room temperature. A volume of 100 µL of Water/Methanol solution (95:5) was added to reconstitute each sample. Samples were then thoroughly mixed by vortex for 10 mins at 5000 rpm and then centrifuged at 16,000 rcf for 4 min at room temperature. The supernatants were transferred to pre-labeled autosampler vials before analysis. LC-MS analysis: The prepared urine samples were analyzed by an LC-MS system comprised of a Waters Acquity UPLC and a Synapt G2Si ESI-Q-TOF mass spectrometer. Chromatographic separation was accomplished on an Acquity HSST3 C18 column (2.1 X 100 mm, 1.8 µm) at 50 °C using a gradient elution comprised by mobile phase A: 0.1% formic acid-water (v/v), and mobile phase B: 0.1% formic acid-methanol (v/v), using a flow rate of 0.400 mL/min. The injection volume was 5 µL.

Chromatography:

Chromatography ID:	CH000666
Chromatography Summary:	Reversed-Phase Gradient Seperation
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity HSS T3 (100 x 2.1mm,1.8um)
Column Pressure:	6,000-10,000 psi
Column Temperature:	50 °C
Flow Rate:	0.4 mL/min
Injection Temperature:	8 °C
Internal Standard:	L-Tryptophan-d5
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Analytical Time:	22 min
Weak Wash Solvent Name:	10% MeOH
Weak Wash Volume:	1000 µL
Strong Wash Solvent Name:	80% MeOH
Strong Wash Volume:	1000 µL
Target Sample Temperature:	8 °C
Sample Loop Size:	10 µL
Sample Syringe Size:	100 µL
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN000934
Analysis Type:	MS
Instrument Name:	Waters Synapt-G2-Si
Chromatography ID:	CH000666
Num Factors:	1
Rt Units:	Minutes
Results File:	ST000610_AN000934_Results.txt
Units:	Normalized Abundance

Analysis ID:	AN000935
Analysis Type:	MS
Instrument Name:	Waters Synapt-G2-Si
Chromatography ID:	CH000666
Num Factors:	1
Rt Units:	Minutes
Results File:	ST000610_AN000935_Results.txt
Units:	Normalized Abundance