Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA045943

Local Sample ID:	C-W74S5-S-02
Subject ID:	SU000851
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000851
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
C-W74S5-S-02	SA045943	FL009130	data	sample data

Collection:

Collection ID:	CO000845
Collection Summary:	All samples stored at -80°C.
Sample Type:	Serum (human), Plasma (human)
Storage Conditions:	-80°C

Treatment:

Treatment ID:	TR000865
Treatment Summary:	N/A

Sample Preparation:

Sampleprep ID:	SP000858
Sampleprep Summary:	Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: External CHEAR Reference Material- Plasma: Parent and Sub-Aliquots: The CHEAR PlasmaRef_20160726 (500 mL) was removed from -80°C storage and thawed by storing it at 4°C overnight. The following day, the thawed plasma was inspected to ensure there was no ice remaining and the bulk plasma 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. 40 mL plasma aliquots were transferred to 50 mL tubes in 4°C room and capped immediately and placed on ice. If needed, the bulk plasma 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. 1 mL plasma aliquots were transferred to 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. Plasma Aliquots for Biocrates platform: The sub-aliquot of CHEAR PlasmaRef_20160726 PP_A_08 was used to prepare aliquots for the Biocrates platform. PP_A_08 was thawed on ice for 30 – 60 min and vortexed briefly on a vortexer, followed by centrifugation at 4°C for 2 minutes at 16,000 rcf. A total of 21 aliquots of 22 µL volumes were prepared as external pool samples for the Biocrates analysis from PP_A_08. These external pool samples were named CHEAR Reference Plasma 1 through 21. Aliquots were stored at -80°C until analysis. CHEAR Christiani Human Cord Blood Serum Samples: Each received sample was transferred from its straw to a new, pre-labeled Lo-bind tube. A volume of 5 µL was transferred from each sample to a new, pre-labeled 7-mL tube to create the total pool sample. The total pool underwent vortex mixing before creating 21 aliquots of 22 µL volumes that were to be used as total pool samples. These total pool samples were named Biocrates Total Pool 1 through 21. Aliquots were stored at -80°C until analysis. Sample preparation for Biocrates Plate: CHEAR Christiani samples, CHEAR Plasma Reference Material external pool samples and Christiani total pool samples were thawed on ice for 30–60 min and placed on ice in the analysis order for sample loading on the p180 Biocrates plate. Biocrates Plate Preparation: A total of three Biocrates plates (1701, 1702, and 1703) were used to accommodate all samples and were prepared following the procedure of AbsoluteIDQ™ p180 Kit. For each plate, the isotope labeled internal standard mix was added to 95 of the 96 wells. The zero sample, QC standards and calibration standards were added to their corresponding wells. A volume of 20 µL of each sample was then added to the appropriate well and dried for 30 minutes under nitrogen flow. All samples were derivatized by adding a 5% solution of phenylisothiocyanate (PITC) in ethanol/pyridine/water (1:1:1, v/v/v) through incubation for 20 min at room temperature and then drying under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all the wells. After shaking and centrifugation through a filter membrane on this plate, 150 µL solution was transferred to a new, 96-well plate and diluted with 150 µL of HPLC grade water for LC-MS analysis to measure amino acids and biogenic amines. The remaining solution in the original plate was diluted with 400 µL of flow injection analysis (FIA) running solvent for FIA-MS analysis to measure lipids, acylcarnitines, and hexose. The Multiple Reaction Monitoring (MRM) mode was used for both LC-MS and FIA-MS analyses.

Sampleprep ID:

SP000858

Sampleprep Summary:

Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: External CHEAR Reference Material- Plasma: Parent and Sub-Aliquots: The CHEAR PlasmaRef_20160726 (500 mL) was removed from -80°C storage and thawed by storing it at 4°C overnight. The following day, the thawed plasma was inspected to ensure there was no ice remaining and the bulk plasma 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. 40 mL plasma aliquots were transferred to 50 mL tubes in 4°C room and capped immediately and placed on ice. If needed, the bulk plasma 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. 1 mL plasma aliquots were transferred to 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. Plasma Aliquots for Biocrates platform: The sub-aliquot of CHEAR PlasmaRef_20160726 PP_A_08 was used to prepare aliquots for the Biocrates platform. PP_A_08 was thawed on ice for 30 – 60 min and vortexed briefly on a vortexer, followed by centrifugation at 4°C for 2 minutes at 16,000 rcf. A total of 21 aliquots of 22 µL volumes were prepared as external pool samples for the Biocrates analysis from PP_A_08. These external pool samples were named CHEAR Reference Plasma 1 through 21. Aliquots were stored at -80°C until analysis. CHEAR Christiani Human Cord Blood Serum Samples: Each received sample was transferred from its straw to a new, pre-labeled Lo-bind tube. A volume of 5 µL was transferred from each sample to a new, pre-labeled 7-mL tube to create the total pool sample. The total pool underwent vortex mixing before creating 21 aliquots of 22 µL volumes that were to be used as total pool samples. These total pool samples were named Biocrates Total Pool 1 through 21. Aliquots were stored at -80°C until analysis. Sample preparation for Biocrates Plate: CHEAR Christiani samples, CHEAR Plasma Reference Material external pool samples and Christiani total pool samples were thawed on ice for 30–60 min and placed on ice in the analysis order for sample loading on the p180 Biocrates plate. Biocrates Plate Preparation: A total of three Biocrates plates (1701, 1702, and 1703) were used to accommodate all samples and were prepared following the procedure of AbsoluteIDQ™ p180 Kit. For each plate, the isotope labeled internal standard mix was added to 95 of the 96 wells. The zero sample, QC standards and calibration standards were added to their corresponding wells. A volume of 20 µL of each sample was then added to the appropriate well and dried for 30 minutes under nitrogen flow. All samples were derivatized by adding a 5% solution of phenylisothiocyanate (PITC) in ethanol/pyridine/water (1:1:1, v/v/v) through incubation for 20 min at room temperature and then drying under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all the wells. After shaking and centrifugation through a filter membrane on this plate, 150 µL solution was transferred to a new, 96-well plate and diluted with 150 µL of HPLC grade water for LC-MS analysis to measure amino acids and biogenic amines. The remaining solution in the original plate was diluted with 400 µL of flow injection analysis (FIA) running solvent for FIA-MS analysis to measure lipids, acylcarnitines, and hexose. The Multiple Reaction Monitoring (MRM) mode was used for both LC-MS and FIA-MS analyses.

Combined analysis:

Analysis ID	AN001311	AN001312	AN001313
Analysis type	MS	MS	MS
Chromatography type	Reversed phase	Isocratic	Isocratic
Chromatography system	Agilent 1100	Agilent 1100	Agilent 1100
Column	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)	no column	no column
MS Type	ESI	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 4000 QTrap	ABI Sciex 4000 QTrap	ABI Sciex 4000 QTrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE
Units	uM	uM	uM

Chromatography:

Chromatography ID:	CH000918
Chromatography Summary:	Reverse Phase Gradient separation
Instrument Name:	Agilent 1100
Column Name:	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)
Column Pressure:	30-120 bar
Column Temperature:	50°C
Flow Rate:	0.5 mL/min
Sample Injection:	10 µL
Target Sample Temperature:	10 °C
Sample Loop Size:	100 µL
Randomization Order:	Yes
Chromatography Type:	Reversed phase

Chromatography ID:	CH000919
Chromatography Summary:	FIA analysis
Instrument Name:	Agilent 1100
Column Name:	no column
Sample Injection:	20 µL
Target Sample Temperature:	10 °C
Sample Loop Size:	100 µL
Randomization Order:	Yes
Chromatography Type:	Isocratic

MS:

MS ID:	MS001204
Analysis ID:	AN001311
Instrument Name:	ABI Sciex 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE
Collision Gas:	Medium
Ion Source Temperature:	500°C
Ion Spray Voltage:	5500

MS ID:	MS001205
Analysis ID:	AN001312
Instrument Name:	ABI Sciex 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE
Collision Gas:	Medium
Ion Spray Voltage:	5500

MS ID:	MS001206
Analysis ID:	AN001313
Instrument Name:	ABI Sciex 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	NEGATIVE
Collision Gas:	Medium
Ion Spray Voltage:	-4500