Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA121623

Local Sample ID:	SCFA_S79
Subject ID:	SU001505
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001505
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SCFA_S79	SA121623	FL014891	Eczema	Treatment

Collection:

Collection ID:	CO001500
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:	TR001520
Treatment Summary:	Control vs Eczema (Non-allergen sensitized atopic eczema, Allergen-sensitiized atopic eczema)

Sample Preparation:

Sampleprep ID:	SP001513
Sampleprep Summary:	500 L of ice-cold extraction solvent (1:1 v/v ACN/water) containing 10 M of d5-benzoic acid as internal standard (IS) was added to 250 mg of wet stool sample and subjected to vortex mixing for 5 min at ambient temperature (24 ± 1 ◦C). The ratio of extraction solvent to wet sample weight was kept constant (2 L:1 mg) to prevent variable extraction efficiencies. The suspension was then centrifuged at 18 000g for 10 min at 4 ◦C. The supernatant was carefully removed and centrifuged again at 18 000g for 10 min at 4 ◦C. An aliquot of 100 L was subsequently derivatized using a final concentration of 10 mM aniline and 5 mM EDC for 2 h at 4 ◦C. The derivatization reaction was quenched using a final concentration of 18 mM succinic acid and 4.6 mM 2-mercaptoethanol for 2 h at 4 ◦C. An aliquot of each sample was further diluted 100-fold. All samples were stored at 4 ◦C until analysis on the same day

Sampleprep ID:

SP001513

Sampleprep Summary:

500 L of ice-cold extraction solvent (1:1 v/v ACN/water) containing 10 M of d5-benzoic acid as internal standard (IS) was added to 250 mg of wet stool sample and subjected to vortex mixing for 5 min at ambient temperature (24 ± 1 ◦C). The ratio of extraction solvent to wet sample weight was kept constant (2 L:1 mg) to prevent variable extraction efficiencies. The suspension was then centrifuged at 18 000g for 10 min at 4 ◦C. The supernatant was carefully removed and centrifuged again at 18 000g for 10 min at 4 ◦C. An aliquot of 100 L was subsequently derivatized using a final concentration of 10 mM aniline and 5 mM EDC for 2 h at 4 ◦C. The derivatization reaction was quenched using a final concentration of 18 mM succinic acid and 4.6 mM 2-mercaptoethanol for 2 h at 4 ◦C. An aliquot of each sample was further diluted 100-fold. All samples were stored at 4 ◦C until analysis on the same day

Combined analysis:

Analysis ID	AN002393
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity
Column	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type	ESI
MS instrument type	Ion trap
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	POSITIVE
Units	nM

Chromatography:

Chromatography ID:	CH001759
Chromatography Summary:	Water and HPLC-grade isopropanol, both acidified with 0.1% formic acid were used as mobile phases A and B respectively. Chromatographic separation was achieved using an Acquity UPLC HSS T3 1.8 M, 2.1 mm x 100 mm column at a flow rate of 0.35 mL/min under the following condition: isocratic 15% B (0.00 − 2.00 min), linear gradient 15% to 33% B (2.01–6.00 min), linear gradient 33% to 34% B (6.01–7.50 min), linear gradient 34% to 36% B (7.51–12.00 min), isocratic 100% B (12.01–13.00 min), isocratic 15% B (13.01–15.00 min). Using an autosampler thermostatted at 4 ◦C, 1 L of each sample was injected onto the column maintained at 50 ◦C. The needle was flushed with methanol postinjection to minimize carry-over effect.
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Flow Gradient:	isocratic 15% B (0.00 - 2.00 min), linear gradient 15% to 33% B (2.01-6.00 min), linear gradient 33% to 34% B (6.01-7.50 min), linear gradient 34% to 36% B (7.51-12.00 min), isocratic 100% B (12.01-13.00 min), isocratic 15% B (13.01-15.00 min).
Flow Rate:	0.35 mL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% isopropanol; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002235
Analysis ID:	AN002393
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Ion trap
MS Type:	ESI
MS Comments:	ion spray voltage, 5500 V; temperature, 500 ◦C; curtain gas, 20 psi; ion source gas 1, 18 psi; and ion source gas 2, 18 psi. Data were acquired in scheduled MRM mode using a 30 s detection window. More details available at Chan, J. C.; Kioh, D. Y.; Yap, G. C.; Lee, B. W.; Chan, E. C., A novel LCMSMS method for quantitative measurement of short-chain fatty acids in human stool derivatized with (12)C- and (13)C-labelled aniline. Journal of pharmaceutical and biomedical analysis 2017, 138, 43-53.
Ion Mode:	POSITIVE