Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA161129

Local Sample ID:	Sulfamethoxypyridazine_24_1_hilicpos_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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Subject:

Subject ID:	SU001792
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	Pooled Liver S9 fractions

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Sulfamethoxypyridazine_24_1_hilicpos_fusion.mzXML	SA161129	FL018986	Sulfamethoxypyridazine	Precursor
Sulfamethoxypyridazine_24_1_hilicpos_fusion.mzXML	SA161129	FL018986	24	Time

Collection:

Collection ID:	CO001785
Collection Summary:	At the specified time point, S9 Reactions were extracted with 2x volume acetonitrile, and spun down at 14,000 rpm for 10 minutes and supernatants were transferred to autosampler vials. Samples were kept at -20°C until instrumental analysis.
Sample Type:	Liver
Storage Conditions:	Described in summary

Treatment:

Treatment ID:	TR001805
Treatment Summary:	Xenobiotic precursors were incubated with pooled human liver S9 fractions with an NADPH regenerating system and other required cofactors and collected at specified time points as described in the associated manuscript.

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

Combined analysis:

Analysis ID	AN002792	AN002793	AN002794	AN002795
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000	Thermo Dionex Ultimate 3000
Column	Waters XBridge BEH Amide (50mm x 2.1mm,2.5um)	Waters XBridge BEH Amide (50mm x 2.1mm,2.5um)	Higgins Analytical Targa C18 (50 x 2.1mm,3um)	Higgins Analytical Targa C18 (50 x 2.1mm,3um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Fusion Tribrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Fusion Tribrid Orbitrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE
Units	relative intensity	relative intensity	relative intensity	relative intensity

Chromatography:

Chromatography ID:	CH002065
Chromatography Summary:	HILIC/ESI+
Chromatography Comments:	HFQE
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters XBridge BEH Amide (50mm x 2.1mm,2.5um)
Chromatography Type:	HILIC

Chromatography ID:	CH002066
Chromatography Summary:	C18/ESI-
Chromatography Comments:	HFQE
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Higgins Analytical Targa C18 (50 x 2.1mm,3um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002588
Analysis ID:	AN002792
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	PRM MSMS, ddMSMS and MS1 data collection, mzmine2
Ion Mode:	POSITIVE

MS ID:	MS002589
Analysis ID:	AN002793
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	ddMSMS and MS1 data collection. mzmine2
Ion Mode:	POSITIVE

MS ID:	MS002590
Analysis ID:	AN002794
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	PRM MSMS, ddMSMS and MS1 data collection. mzmine2
Ion Mode:	NEGATIVE

MS ID:	MS002591
Analysis ID:	AN002795
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	ddMSMS and MS1 data collection. mzmine2
Ion Mode:	NEGATIVE