Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA019421

Local Sample ID:	Case_70056
Subject ID:	SU000426
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	female
Species Group:	Human

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Subject:

Subject ID:	SU000426
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	female
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Case_70056	SA019421	FL004740	PA	Group
Case_70056	SA019421	FL004740	N	Vaginal Bleeding

Collection:

Collection ID:	CO000420
Collection Summary:	Serum samples collected in the second trimester from pregnant women
Sample Type:	Blood
Blood Serum Or Plasma:	serum

Treatment:

Treatment ID:	TR000440
Treatment Summary:	no treatment

Sample Preparation:

Sampleprep ID:	SP000433
Sampleprep Summary:	Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: Serum samples were thawed on ice and pooled QC samples were prepared. Samples (study and pooled QC samples) were then diluted, vortexed, and transferred into properly labeled 2 mL Lo-Bind eppendorf tubes and dried on a lyophilizer overnight. The residue was reconstituted with 30 µL of PBS buffer. Samples were then vortexed and then centrifuged at room temperature and at 16,000 rcf for 4.0 minutes. All study samples and pooled QC samples were splitted into two AbsoluteIDQ p180 Kit-96 wells plates (Plate 1502 and 1503). Biocrates Plate Preparation: The Biocrates p180 kit-96 wells plates were prepared following the AbsoluteIDQ p180 Kit metabolomics procedure. Briefly, an internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC standards and calibration standards were added to their corresponding wells. The study samples and pooled QC Samples were then added to the appropriate wells and dried for 30 minutes under nitrogen flow. The plate was derivatized using a 5% Phenylisothiocyanate (PITC) solution in (1:1:1) Ethanol:Pyridine:Water and, then, incubated for 20 minutes followed by a drying step under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. The plate was then vortexed and filtrated by centrifugation. After centrifugation, 150 µL was removed and transferred to a second 96-well plate (LC-MS/MS plate). This second plate was diluted with 150 µL of HPLC grade water for a subsequent LC-MS/MS assay (analysis of amino acids and biogenic amines). All the wells in the original plate was diluted with 400 µL of flow injection analysis (FIA) Running Solvent for a FIA-MS analysis (lipids, acylcarnitines, and hexose analysis).

Sampleprep ID:

SP000433

Sampleprep Summary:

Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: Serum samples were thawed on ice and pooled QC samples were prepared. Samples (study and pooled QC samples) were then diluted, vortexed, and transferred into properly labeled 2 mL Lo-Bind eppendorf tubes and dried on a lyophilizer overnight. The residue was reconstituted with 30 µL of PBS buffer. Samples were then vortexed and then centrifuged at room temperature and at 16,000 rcf for 4.0 minutes. All study samples and pooled QC samples were splitted into two AbsoluteIDQ p180 Kit-96 wells plates (Plate 1502 and 1503). Biocrates Plate Preparation: The Biocrates p180 kit-96 wells plates were prepared following the AbsoluteIDQ p180 Kit metabolomics procedure. Briefly, an internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC standards and calibration standards were added to their corresponding wells. The study samples and pooled QC Samples were then added to the appropriate wells and dried for 30 minutes under nitrogen flow. The plate was derivatized using a 5% Phenylisothiocyanate (PITC) solution in (1:1:1) Ethanol:Pyridine:Water and, then, incubated for 20 minutes followed by a drying step under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. The plate was then vortexed and filtrated by centrifugation. After centrifugation, 150 µL was removed and transferred to a second 96-well plate (LC-MS/MS plate). This second plate was diluted with 150 µL of HPLC grade water for a subsequent LC-MS/MS assay (analysis of amino acids and biogenic amines). All the wells in the original plate was diluted with 400 µL of flow injection analysis (FIA) Running Solvent for a FIA-MS analysis (lipids, acylcarnitines, and hexose analysis).

Combined analysis:

Analysis ID	AN000645
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1100
Column	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex API 4000 QTrap
Ion Mode	UNSPECIFIED
Units	area

Chromatography:

Chromatography ID:	CH000469
Chromatography Summary:	Biocrates P180 analysis combines a RP for positive mode MS , a flow injection for both pos and neg mode MS
Instrument Name:	Agilent 1100
Column Name:	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)
Column Pressure:	30-120 bar for RP and 4-50 bar for FIA
Column Temperature:	50 c fro RP and RT for FIA
Flow Rate:	0.5 ml/min for RP and 0.2 ml/min for FIA
Sample Injection:	10 ul
Solvent A:	100% water; 0.2% formic acid
Solvent B:	100% acetonitrile; 0.2% formic acid for RP; 100% methanol with Biocrates FIa Solvent
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000577
Analysis ID:	AN000645
Instrument Name:	ABI Sciex API 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Biocrates combines a pos and negative mode for analysis
Ion Mode:	UNSPECIFIED
Analysis Protocol File:	KIT2-LC-5404