Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA317548

Local Sample ID:	Con_39
Subject ID:	SU003036
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	infant
Gender:	Not applicable
Human Race:	Pacific asian

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Subject:

Subject ID:	SU003036
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	infant
Gender:	Not applicable
Human Race:	Pacific asian

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Con_39	SA317548	FL037816	Con	Factor

Collection:

Collection ID:	CO003029
Collection Summary:	The study population consisted of 158 six-month-old infants (46 healthy infants, 30 only AD group, and 82 with combined AD and FA) involved in the Cohort for Childhood Origin of Asthma and Allergic Diseases (COCOA) (reference: BMC Pulm Med 2014, 14:109.)
Sample Type:	Feces

Treatment:

Treatment ID:	TR003045
Treatment Summary:	no treatment

Sample Preparation:

Sampleprep ID:	SP003042
Sampleprep Summary:	~10 mg of the freeze-dried feces was homogenized well with an internal standard solution (200 μl of 1 μM C17 S1P solution). Then, 300 μl of chloroform, 200 μl of methanol, 50 μl of H2O, and 10 μl of 10N NaOH were added to each solution, and vortex well. The aqueous layer was collected after centrifugation, where S1P was separated from other lipids under alkaline condition. 50 uL of H2O was added to the remaining solution and the aqueous layer was collected again after centrifugation. Then, S1P was re-extracted into the chloroform phase under acidic conditions using 400 μl of chloroform and 20 μl of 10M HCl. This step was repeated three times. The chloroform was evaporated, and the dried samples were reconstituted with methanol prior to LC-MS/MS analysis.

Sampleprep ID:

SP003042

Sampleprep Summary:

~10 mg of the freeze-dried feces was homogenized well with an internal standard solution (200 μl of 1 μM C17 S1P solution). Then, 300 μl of chloroform, 200 μl of methanol, 50 μl of H2O, and 10 μl of 10N NaOH were added to each solution, and vortex well. The aqueous layer was collected after centrifugation, where S1P was separated from other lipids under alkaline condition. 50 uL of H2O was added to the remaining solution and the aqueous layer was collected again after centrifugation. Then, S1P was re-extracted into the chloroform phase under acidic conditions using 400 μl of chloroform and 20 μl of 10M HCl. This step was repeated three times. The chloroform was evaporated, and the dried samples were reconstituted with methanol prior to LC-MS/MS analysis.

Combined analysis:

Analysis ID	AN004793
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000
Column	Phenomenex Jupiter C4 (50 × 1.0 mm, 5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo LTQ XL
Ion Mode	NEGATIVE
Units	pmol/mg

Chromatography:

Chromatography ID:	CH003624
Chromatography Summary:	Ultimate 3000-gradient
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Phenomenex Jupiter C4 (50 × 1.0 mm, 5um)
Column Temperature:	35
Flow Gradient:	10% B for 0 min, 10–90% B for 5 min, 90% B for 10 min, 90–10% B for 0.1 min, and 10% B for 4.9 min
Flow Rate:	300 μL/min
Solvent A:	0.1% formic acid in H2O
Solvent B:	0.1% formic acid in MeOH
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004539
Analysis ID:	AN004793
Instrument Name:	Thermo LTQ XL
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Selected ion monitoring (SRM) was uesd in the negative ion mode and the extracted ion chromatogram corresponding to the specific transition (phosphate ion, PO3-, m/z 78.9585) for S1P was used for quantification. Calibration range was 0.01 – 10 μM with R2 > 0.99. Data analysis was performed by using Xcalibur 2.2 software. Ultimate3000
Ion Mode:	NEGATIVE