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MB Sample ID: SA215410

Local Sample ID:9337
Subject ID:SU002333
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

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Sample Preparation:

Sampleprep ID:SP002339
Sampleprep Summary:Stool samples were homogenized in 10 μL of water per milligram stool sample weight using a bead mill (TissueLyser II; Qiagen) and the aqueous homogenates were aliquoted for metabolite profiling analyses. LC-MS samples were prepared for four profiling methods as follows: HILIC-pos: Metabolites were extracted by adding 90 μL of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C8-pos: Lipids were extracted by adding 190 μL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, ambient temperature) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. HILIC-neg: Metabolites were extracted by adding 120 μL of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C18-neg: Metabolites were extracted by adding 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis.
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