Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA023202

Local Sample ID:	S54
Subject ID:	SU000478
Subject Type:	Animal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammal

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Sample Preparation:

Sampleprep ID:	SP000485
Sampleprep Summary:	Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: Thawed serum samples were vortexed for 30 seconds. Whole study pooled QC samples were created by combining 10 µL aliquot from each of the study samples into a 2 mL LoBind eppendorf tube. This QC pooled sample was then vortexed for 30 sec. Then, three whole study pooled QC samples of 30 µL each were aliquoted into 2 mL LoBind eppendorf tubes. Study samples were generated by aliquoting 30 µL from each original sample vial into 2 mL LoBind eppendorf tubes. For extraction, 1,000 µL of cold 3:3:2 Acetonitrile:Isopropyl Alcohol:Water (v/v/v) was added to each tube and vortexed for 5 min at 4 °C. The samples were then centrifuged at 4 °C and at 14,000 rcf for 2 min. A 450 µL aliquot of the supernatant from each sample was transferred into pre-labeled 2.0 mL LoBind eppendorf tubes and stored at -80 °C. Samples were then dried on a lyophilizer overnigh. The residue was reconstituted in 30 µL of 85:15 Ethanol:Water, v/v, and vortexed. Then, the samples were centrifuged at 4 °C for 4 min at 16,000 rcf. Biocrates Plate Preparation: A Biocrates p180 kit was prepared following the AbsoluteIDQ™ p180 Kit metabolomics procedure. Briefly, an internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC standards and calibration standards were added to their corresponding wells. The study samples and pooled QC Samples (20 µL) were then added to the appropriate wells and dried for 30 minutes under nitrogen flow. The plate was derivatized using a 5% phenylisothiocyanate (PITC) solution in (1:1:1) ethanol:pyridine:water (v/v/v) and, then, incubated for 20 minutes followed by a drying step under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. The plate was then shaken and centrifuged. After centrifugation, 150 µL was removed and transferred to a second 96-well plate (LCMS plate). This second plate was diluted with 150 µL of HPLC grade water for a subsequent LCMS (MRM analysis) for measuring amino acids and biogenic amines. All wells in the original plate were diluted with 400 µL of flow injection analysis (FIA) Running Solvent for a FIA-MS (MRM analysis) for measuring lipids, acylcarnitines, and hexose.

Sampleprep ID:

SP000485

Sampleprep Summary:

Sample Preparation Prior to Biocrates p180 Kit Plate Analysis: Thawed serum samples were vortexed for 30 seconds. Whole study pooled QC samples were created by combining 10 µL aliquot from each of the study samples into a 2 mL LoBind eppendorf tube. This QC pooled sample was then vortexed for 30 sec. Then, three whole study pooled QC samples of 30 µL each were aliquoted into 2 mL LoBind eppendorf tubes. Study samples were generated by aliquoting 30 µL from each original sample vial into 2 mL LoBind eppendorf tubes. For extraction, 1,000 µL of cold 3:3:2 Acetonitrile:Isopropyl Alcohol:Water (v/v/v) was added to each tube and vortexed for 5 min at 4 °C. The samples were then centrifuged at 4 °C and at 14,000 rcf for 2 min. A 450 µL aliquot of the supernatant from each sample was transferred into pre-labeled 2.0 mL LoBind eppendorf tubes and stored at -80 °C. Samples were then dried on a lyophilizer overnigh. The residue was reconstituted in 30 µL of 85:15 Ethanol:Water, v/v, and vortexed. Then, the samples were centrifuged at 4 °C for 4 min at 16,000 rcf. Biocrates Plate Preparation: A Biocrates p180 kit was prepared following the AbsoluteIDQ™ p180 Kit metabolomics procedure. Briefly, an internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC standards and calibration standards were added to their corresponding wells. The study samples and pooled QC Samples (20 µL) were then added to the appropriate wells and dried for 30 minutes under nitrogen flow. The plate was derivatized using a 5% phenylisothiocyanate (PITC) solution in (1:1:1) ethanol:pyridine:water (v/v/v) and, then, incubated for 20 minutes followed by a drying step under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. The plate was then shaken and centrifuged. After centrifugation, 150 µL was removed and transferred to a second 96-well plate (LCMS plate). This second plate was diluted with 150 µL of HPLC grade water for a subsequent LCMS (MRM analysis) for measuring amino acids and biogenic amines. All wells in the original plate were diluted with 400 µL of flow injection analysis (FIA) Running Solvent for a FIA-MS (MRM analysis) for measuring lipids, acylcarnitines, and hexose.