Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA121685

Local Sample ID:	Global_S147
Subject ID:	SU001506
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001506
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Global_S147	SA121685	FL014892	Control	Treatment

Collection:

Collection ID:	CO001501
Collection Summary:	Stool samples were collected by the parents using sterile faeces containers and stored at -20oC at home. Samples were then transported to the lab in cold chain within 20 hours of sample collection for processing. After processing, sample were stored at -80oC until further analysis.
Sample Type:	Feces
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001521
Treatment Summary:	Control vs Eczema (Non-allergen sensitized atopic eczema, Allergen-sensitiized atopic eczema)

Sample Preparation:

Sampleprep ID:	SP001514
Sampleprep Summary:	For global profiling of stool metabolites, samples were randomized then processed according to our in-house protocol. Briefly, lyophilized stool sample (80 mg) was ultrasonicated with 1 mL of ice-cold extraction solvent (methanol:water [8:2]) containing 1 µg/mL d27-myristic acid as an internal standard at 4°C in a bath ultrasonicator (Elma Transsonic 460, Germany) for 30 min, and vortex-mixed for 2 min. The samples were then centrifuged at 18,000 g for 20 min at 4°C, and 0.5 mL of the supernatant was extracted carefully followed by drying at 50°C under a gentle stream of nitrogen gas (Turbovap LV, Caliper Life Sciences, Hopkinton, MA, USA). 100 µL of toluene (kept anhydrous with sodium sulfate) was added to the dry residue and evaporated completely again at 50°C under nitrogen gas to remove traces of water. The dried metabolic extract was then oximated with 50 µL of MOX (20 mg/mL) at 60°C for 2 h. Following centrifugation, 100 µL of MSTFA with 1% TMCS was added and the mixture was incubated at 60°C for 45 min to form trimethylsilyl (TMS) derivatives. Finally, 100 µL of the TMS derivatives was transferred into a GC vial and subjected to gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) analysis. 50 µL of each stool extract prepared during extraction of lyophilized stool were also pooled to prepare quality control (QC) samples.

Sampleprep ID:

SP001514

Sampleprep Summary:

For global profiling of stool metabolites, samples were randomized then processed according to our in-house protocol. Briefly, lyophilized stool sample (80 mg) was ultrasonicated with 1 mL of ice-cold extraction solvent (methanol:water [8:2]) containing 1 µg/mL d27-myristic acid as an internal standard at 4°C in a bath ultrasonicator (Elma Transsonic 460, Germany) for 30 min, and vortex-mixed for 2 min. The samples were then centrifuged at 18,000 g for 20 min at 4°C, and 0.5 mL of the supernatant was extracted carefully followed by drying at 50°C under a gentle stream of nitrogen gas (Turbovap LV, Caliper Life Sciences, Hopkinton, MA, USA). 100 µL of toluene (kept anhydrous with sodium sulfate) was added to the dry residue and evaporated completely again at 50°C under nitrogen gas to remove traces of water. The dried metabolic extract was then oximated with 50 µL of MOX (20 mg/mL) at 60°C for 2 h. Following centrifugation, 100 µL of MSTFA with 1% TMCS was added and the mixture was incubated at 60°C for 45 min to form trimethylsilyl (TMS) derivatives. Finally, 100 µL of the TMS derivatives was transferred into a GC vial and subjected to gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) analysis. 50 µL of each stool extract prepared during extraction of lyophilized stool were also pooled to prepare quality control (QC) samples.

Combined analysis:

Analysis ID	AN002394
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890A
Column	Agilent DB-1 DB-1 (30 m × 250 um i.d.)
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Leco Pegasus III GC TOF
Ion Mode	POSITIVE
Units	Ion Count Per Second

Chromatography:

Chromatography ID:	CH001760
Chromatography Summary:	Helium was employed as the carrier gas at a constant flow rate of 1.5 mL/min and the injector split ratio was adjusted to 1:10. An injection volume of 1 µL was used. The injector, transfer line and ionsource temperatures were maintained at 220, 280 and 250°C, respectively. The oven temperature was programmed at 70°C for 0.2 min, increased at 9°C/min to 270°C where it was sustained for 5 min, and further increased at 40°C/min to 310°C where it was held for 11 min. The mass spectrometer was operated in the electron impact ionization mode at 70 eV. Data were acquired in full scan mode from m/z 40 to 600 with an acquisition rate of 20 spectra per second. To detect retention time shifts and facilitate Kovats retention index (RI) calculation, a standard alkane series mixture (C7-C30) was injected before every batch during sample analysis. This was accompanied by an injection of one pooled QC after every injection of 10 samples.
Instrument Name:	Agilent 7890A
Column Name:	Agilent DB-1 DB-1 (30 m × 250 um i.d.)
Chromatography Type:	GC

MS:

MS ID:	MS002236
Analysis ID:	AN002394
Instrument Name:	Leco Pegasus III GC TOF
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	The mass spectrometer was operated in the electron impact ionization mode at 70 eV. Data were acquired in full scan mode from m/z 40 to 600 with an acquisition rate of 20 spectra per second.
Ion Mode:	POSITIVE