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MB Sample ID: SA186165

Local Sample ID:	5005933
Subject ID:	SU002072
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Sample Preparation:

Sampleprep ID:	SP002078
Sampleprep Summary:	Briefly, 20 μL of serum (prepared by adding 1:20 (m/v) ultrapure water to 50 mg of feces) was filtered through a Ostro Protein Precipitation and Phospholipid Removal 96-well plate (Waters Corporation, Milford, USA), using 100 μL of cold methanol contemning the internal standard mixtures (LCA-d4, TCA-d4, GUDCA-d4, GCA-d4, CA-d4, UDCA-d4, GCDCA-d4, CDCA-d4, DCA-d4, GLCA-d4). The eluent was collected and evaporated to dryness and the residue was re-suspended in 20 μL of a 40:60 MeOH: H2O v/v mixture. The analyses were performed on an ACQUITY HSS T3 (2.1×100 mm, 1.8 μm) column, Waters (Milford), coupled to a triple quadrupole mass spectrometer (Waters Corporation, Milford, USA) with an atmospheric electrospray interface operating in negative ion mode. Separation was performed using gradient elution with 0.1 % formic acid in water (v/v) (A) and 0.1 % formic acid in acetonitrile:methanol (3:1, v/v) (B) at a flow rate of 0.5 mL/ min. Gradient program was 0 min 15 % B, 0-1 min; 30 % B, 1- 16 min; 16-18 min; 70 % B, 18–23 min 100 % B, and equilibrium time between runs was 7 min. The injection volume was 5 μL and the column was kept at 35 °C. An external calibration with nine calibration points (0.0025–600 ng/mL) was carried out for use in quantitation.

Sampleprep ID:

SP002078

Sampleprep Summary:

Briefly, 20 μL of serum (prepared by adding 1:20 (m/v) ultrapure water to 50 mg of feces) was filtered through a Ostro Protein Precipitation and Phospholipid Removal 96-well plate (Waters Corporation, Milford, USA), using 100 μL of cold methanol contemning the internal standard mixtures (LCA-d4, TCA-d4, GUDCA-d4, GCA-d4, CA-d4, UDCA-d4, GCDCA-d4, CDCA-d4, DCA-d4, GLCA-d4). The eluent was collected and evaporated to dryness and the residue was re-suspended in 20 μL of a 40:60 MeOH: H2O v/v mixture. The analyses were performed on an ACQUITY HSS T3 (2.1×100 mm, 1.8 μm) column, Waters (Milford), coupled to a triple quadrupole mass spectrometer (Waters Corporation, Milford, USA) with an atmospheric electrospray interface operating in negative ion mode. Separation was performed using gradient elution with 0.1 % formic acid in water (v/v) (A) and 0.1 % formic acid in acetonitrile:methanol (3:1, v/v) (B) at a flow rate of 0.5 mL/ min. Gradient program was 0 min 15 % B, 0-1 min; 30 % B, 1- 16 min; 16-18 min; 70 % B, 18–23 min 100 % B, and equilibrium time between runs was 7 min. The injection volume was 5 μL and the column was kept at 35 °C. An external calibration with nine calibration points (0.0025–600 ng/mL) was carried out for use in quantitation.