Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA160952

Local Sample ID:	PipemidicAcid_24_2_c18neg_fusion.mzXML
Subject ID:	SU001792
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	Pooled Liver S9 fractions

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Sample Preparation:

Sampleprep ID:	SP001798
Sampleprep Summary:	Each test compound stock solution was diluted to 0.3 mM with water prior to incubation with S9 enzymes. The NADPH regenerating system was reconstituted with addition of 3.5 ml of water to make a nal volume of 5 ml. Cofactors were combined to form a 4X cofactor stock as follows, prior to addition into the reaction mixture: 10 mM UDPGA, 2 mM GSH, 2 mg/ml PAPS, 0.1 mM acetyl-CoA, and NADPH regenerating system (1 mM NADP, 5 mM glucose-6-phosphate, 1 unit glucose-6-phosphate dehydrogenase). Reactions were carried out at 30 °C on 96-well plates (Fig. 1). S9 fraction was diluted 10-fold in water immediately before mixing with 0.2 M Tris-Cl, pH 7.5/2 mM MgCl2 and 0.3 mM of the xenobiotic solution in a 1:1:1 ratio (15 µL each). and incubated at 30 °C for 5 min. To start the reaction, 15 µL of 4X cofactor stock was added, and incubation was carried out at 30 °C for the indicated times. To terminate the reaction, we added a three-fold volume of acetonitrile, covered the plate with paralm, vortexed, and froze at − 20 °C to precipitate insoluble materials such as protein. After thawing and centrifugation of the incubation plate, the supernatants were transferred into polypropylene autosampler vials, which were stored at − 20 °C until instrumental analysis

Sampleprep ID:

SP001798

Sampleprep Summary:

Each test compound stock solution was diluted to 0.3 mM with water prior to incubation with S9 enzymes. The NADPH regenerating system was reconstituted with addition of 3.5 ml of water to make a nal volume of 5 ml. Cofactors were combined to form a 4X cofactor stock as follows, prior to addition into the reaction mixture: 10 mM UDPGA, 2 mM GSH, 2 mg/ml PAPS, 0.1 mM acetyl-CoA, and NADPH regenerating system (1 mM NADP, 5 mM glucose-6-phosphate, 1 unit glucose-6-phosphate dehydrogenase). Reactions were carried out at 30 °C on 96-well plates (Fig. 1). S9 fraction was diluted 10-fold in water immediately before mixing with 0.2 M Tris-Cl, pH 7.5/2 mM MgCl2 and 0.3 mM of the xenobiotic solution in a 1:1:1 ratio (15 µL each). and incubated at 30 °C for 5 min. To start the reaction, 15 µL of 4X cofactor stock was added, and incubation was carried out at 30 °C for the indicated times. To terminate the reaction, we added a three-fold volume of acetonitrile, covered the plate with paralm, vortexed, and froze at − 20 °C to precipitate insoluble materials such as protein. After thawing and centrifugation of the incubation plate, the supernatants were transferred into polypropylene autosampler vials, which were stored at − 20 °C until instrumental analysis