Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA172804

Local Sample ID:	CE089
Subject ID:	SU001928
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001928
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
CE089	SA172804	FL020831	Centenarian	Category

Collection:

Collection ID:	CO001921
Collection Summary:	Stool samples were collected by the individuals using sterile faeces containers and transported to the lab in cooling-box with ice and Anaero pack within 24 hours of sample collection. Immediately after arriving, samples were aliquoted and stored at -80°C until further analysis.
Sample Type:	Feces
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001940
Treatment Summary:	Frozen feces were crushed with a metal bead using a Multi-Beads Shocker (Yasui Kikai).

Sample Preparation:

Sampleprep ID:	SP001934
Sampleprep Summary:	Accurately weighed freeze-crushed faecal samples were resuspended in 20 times the volume of water (e.g. 12.5 mg of faece in 250 μL water). 250 μL of faecal suspension was homogenized in 747 μL of 0.27 N NaOH by ultrasonication for 1 hr in a screw-cap glass vial containing 3 μL of deuterium-labelled internal standards (d4-CA, d4-GCDCA, d4-TCDCA, d4-CDCA-3S, and d4-LCA, 50 μM each). After incubation for 1 hr at room temperature, pH was adjusted to 8.0 using 8 N HCl, mixed with 110 μL of 0.5 M EDTA/0.5 M TrisHCl. The solution was centrifuged at 15,000 rpm and the supernatant was transferred onto a solid-phase extraction cartridge (Agilent Bond Elut C18, 100 mg/3 mL, preconditioned with 1 mL of methanol and 3 mL of water, three times). The cartridge was washed with 1 mL of water and captured bile acids were eluted with 300 μL of 90% ethanol.

Sampleprep ID:

SP001934

Sampleprep Summary:

Accurately weighed freeze-crushed faecal samples were resuspended in 20 times the volume of water (e.g. 12.5 mg of faece in 250 μL water). 250 μL of faecal suspension was homogenized in 747 μL of 0.27 N NaOH by ultrasonication for 1 hr in a screw-cap glass vial containing 3 μL of deuterium-labelled internal standards (d4-CA, d4-GCDCA, d4-TCDCA, d4-CDCA-3S, and d4-LCA, 50 μM each). After incubation for 1 hr at room temperature, pH was adjusted to 8.0 using 8 N HCl, mixed with 110 μL of 0.5 M EDTA/0.5 M TrisHCl. The solution was centrifuged at 15,000 rpm and the supernatant was transferred onto a solid-phase extraction cartridge (Agilent Bond Elut C18, 100 mg/3 mL, preconditioned with 1 mL of methanol and 3 mL of water, three times). The cartridge was washed with 1 mL of water and captured bile acids were eluted with 300 μL of 90% ethanol.

Combined analysis:

Analysis ID	AN002999
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Exion LC
Column	InertSustain C18
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex Triple Quad 6500+
Ion Mode	UNSPECIFIED
Units	nM

Chromatography:

Chromatography ID:	CH002224
Chromatography Summary:	2 μL was injected to LC/ESI-MS/MS (Triple Quad 6500+ tandem mass spectrometer, equipped with an ESI probe and Exion LC AD ultra-high pressure liquid chromatography system; SCIEX). A separation column, InertSustain C18 (150 mm × 2.1 mm ID, 3 μm particle size; GL Sciences Inc.), was utilized at 40 °C. A mixture of 10 mM ammonium acetate and acetonitrile was used as the eluent and the separation was carried out by linear gradient elution at a flow rate of 0.2 mL/min. The mobile phase composition was gradually changed as follows: ammonium acetate-acetonitrile (86:14, v/v) for 0.5 min, (78:22, v/v) for 0.5-5 min, (72:28, v/v) for 5-28 min, (46:54, v/v) for 28-55 min, (2:98, v/v) for 55-66 min, and (2:98, v/v) for 4 min. The total run time was 70 min.
Instrument Name:	Exion LC
Column Name:	InertSustain C18
Column Temperature:	40
Flow Gradient:	ammonium acetate-acetonitrile (86:14, v/v) for 0.5 min, (78:22, v/v) for 0.5-5 min, (72:28, v/v) for 5-28 min, (46:54, v/v) for 28-55 min, (2:98, v/v) for 55-66 min, and (2:98, v/v) for 4 min. The total run time was 70 min.
Flow Rate:	0.2 mL/min
Solvent A:	100% water; 10mM ammonium acetate
Solvent B:	100% acetonitrile
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002788
Analysis ID:	AN002999
Instrument Name:	ABI Sciex Triple Quad 6500+
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The following MS parameters were used for the positive ion MRM mode: ion spray voltage; 5,500 V, interface temperature; 500 °C, curtain gas; 30 psi, collision gas (nitrogen); 9 psi, nebulizing gas; 80 psi, and heater gas; 80 psi. For the negative ion MRM mode: ion spray voltage; -4,500 V, interface temperature; 500 °C, curtain gas; 30 psi, collision gas (nitrogen); 9 psi, nebulizing gas; 80 psi, and heater gas; 80 psi. Samples were obtained using Analyst software ver1.71 and analysed using SCIEX OS-MQ software ver1.7.0.36606.
Ion Mode:	UNSPECIFIED