Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA010747

Local Sample ID:	MAL1
Subject ID:	SU000241
Subject Type:	Animal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Age Or Age Range:	6 weeks at collection
Gender:	Female
Animal Animal Supplier:	Jackson Labs
Animal Housing:	Conventional
Animal Feed:	Low Protein, Low Fat Malnourished Diet/Isocaloric Control Diet
Cell Primary Immortalized:	Malnourished/Control
Species Group:	Mammal

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Subject:

Subject ID:	SU000241
Subject Type:	Animal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Age Or Age Range:	6 weeks at collection
Gender:	Female
Animal Animal Supplier:	Jackson Labs
Animal Housing:	Conventional
Animal Feed:	Low Protein, Low Fat Malnourished Diet/Isocaloric Control Diet
Cell Primary Immortalized:	Malnourished/Control
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
MAL1	SA010747	FL002567	malnourished	Diet

Collection:

Collection ID:	CO000229
Collection Summary:	-
Sample Type:	Small intestinal fecal contents
Collection Time:	6 weeks of age
Storage Conditions:	-80 degrees
Tissue Cell Quantity Taken:	15-40mg

Treatment:

Treatment ID:	TR000249
Treatment Summary:	4 mice were given a malnourished diet and 4 mice were untreated, fed a control diet

Sample Preparation:

Sampleprep ID:	SP000243
Sampleprep Summary:	Bile-acid targeted metabolomics. Each sample was homogenized in LC-MS grade water at a ratio of 150 ?L per 10 mg raw material and with the aid of 5-mm stainless steel metal balls. Bile acids were extracted by addition of acetonitrile at a ratio of 350 ?L per 10 mg raw material followed by vortexing and sonication (1 min) in an ice-water ultrasonic bath. The samples were centrifuged. 20 ?L of the supernatants were precisely taken out and mixed with a predefined mix of 14 deuterium-labeled bile acids as the internal standards. The mixtures were subjected to phospholipid-depletion solid-phase extraction according to a validated protocol for sample cleanup and bile acid enrichment62. The flow-through fractions were collected and then dried under a gentle nitrogen flow. The dried residues were dissolved in 200 ?L of 50% methonal. 10 ?L were injected for quantitation by UPLC-MRM/MS. A Dionex UPLC system was connected to an AB Sciex 4000 QTRAP mass spectrometer which was operated in the negative ion multiple-reaction monitoring (MRM) mode and with electrospray ionization. UPLC separation was carried out on a 15 cm long C-18 UPLC column with water-acetonitrile-formic acid as the mobile phase for binary gradient elution using a developed and validated protocol for comprehensive analysis of bile acids in biological samples (Han, etc. manuscript submitted to Analytical Chemistry). The column temperature was 45 oC and the flow rate was 0.35 mL/min. 45 bile acids (including the 19 targeted bile acids) were involved in the quantitation by UPLC/scheduled MRM/MS. Concentrations of the detected bile acids were calculated with internal standard calibration from the linearly regressed standard calibration curves of individual bile acids. The lower limits of quantitation were 0.08 nmoles/mg for all the bile acids.

Sampleprep ID:

SP000243

Sampleprep Summary:

Bile-acid targeted metabolomics. Each sample was homogenized in LC-MS grade water at a ratio of 150 ?L per 10 mg raw material and with the aid of 5-mm stainless steel metal balls. Bile acids were extracted by addition of acetonitrile at a ratio of 350 ?L per 10 mg raw material followed by vortexing and sonication (1 min) in an ice-water ultrasonic bath. The samples were centrifuged. 20 ?L of the supernatants were precisely taken out and mixed with a predefined mix of 14 deuterium-labeled bile acids as the internal standards. The mixtures were subjected to phospholipid-depletion solid-phase extraction according to a validated protocol for sample cleanup and bile acid enrichment62. The flow-through fractions were collected and then dried under a gentle nitrogen flow. The dried residues were dissolved in 200 ?L of 50% methonal. 10 ?L were injected for quantitation by UPLC-MRM/MS. A Dionex UPLC system was connected to an AB Sciex 4000 QTRAP mass spectrometer which was operated in the negative ion multiple-reaction monitoring (MRM) mode and with electrospray ionization. UPLC separation was carried out on a 15 cm long C-18 UPLC column with water-acetonitrile-formic acid as the mobile phase for binary gradient elution using a developed and validated protocol for comprehensive analysis of bile acids in biological samples (Han, etc. manuscript submitted to Analytical Chemistry). The column temperature was 45 oC and the flow rate was 0.35 mL/min. 45 bile acids (including the 19 targeted bile acids) were involved in the quantitation by UPLC/scheduled MRM/MS. Concentrations of the detected bile acids were calculated with internal standard calibration from the linearly regressed standard calibration curves of individual bile acids. The lower limits of quantitation were 0.08 nmoles/mg for all the bile acids.

Combined analysis:

Analysis ID	AN000331
Analysis type	MS
Chromatography type
Chromatography system
Column
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex API 4000 QTrap
Ion Mode	NEGATIVE
Units	nM/mg

Chromatography:

Chromatography ID:	CH000248

Chromatography ID:

CH000248

MS:

MS ID:	MS000280
Analysis ID:	AN000331
Instrument Name:	ABI Sciex API 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	NEGATIVE