Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA271211

Local Sample ID:	pARDS_60_S1P
Subject ID:	SU002807
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002813
Sampleprep Summary:	Sample solutions were prepared by using commonly used liquid-liquid extaction procedure known as Bligh/Dyer mthod with minor modifications.(Can.J.Biochem.Physiol. 37:911-917) Briefly, 400 μL of chloroform/methanol (1/2, v/v) was added to each sample solution and mixed well. The solution was centrifuged for 15 min at 14000 rpm. After centrifugation, a thick precipitate containing macromolecules was found between the aqueous upper layer and the organic lower layer. Polar metabolites were contained in the upper aqueous phase. The collected volume from each layer were generally the same, however sometimes any specific sample had thicker interface between organic and aqueous layer, which resulted in little variation of the recovered volumes. However, internal standards added prior to sample preparation should correct this variation. The aqueous phases were dried under vacuum and stored at -20℃ until further analysis. The dried matter from the aqueous solutions was reconstituted with 50 μL of H2O/MeOH (50/50 v/v) prior to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. For amino acids, 10 μL out of the total 50 μL reconstituted aqueous phase was used for chemical derivatization of amino acids using phenylisothiocyanate.For S1P and free fatty acids, separate serum samples were used. Details can be found in attached method file.

Sampleprep ID:

SP002813

Sampleprep Summary:

Sample solutions were prepared by using commonly used liquid-liquid extaction procedure known as Bligh/Dyer mthod with minor modifications.(Can.J.Biochem.Physiol. 37:911-917) Briefly, 400 μL of chloroform/methanol (1/2, v/v) was added to each sample solution and mixed well. The solution was centrifuged for 15 min at 14000 rpm. After centrifugation, a thick precipitate containing macromolecules was found between the aqueous upper layer and the organic lower layer. Polar metabolites were contained in the upper aqueous phase. The collected volume from each layer were generally the same, however sometimes any specific sample had thicker interface between organic and aqueous layer, which resulted in little variation of the recovered volumes. However, internal standards added prior to sample preparation should correct this variation. The aqueous phases were dried under vacuum and stored at -20℃ until further analysis. The dried matter from the aqueous solutions was reconstituted with 50 μL of H2O/MeOH (50/50 v/v) prior to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. For amino acids, 10 μL out of the total 50 μL reconstituted aqueous phase was used for chemical derivatization of amino acids using phenylisothiocyanate.For S1P and free fatty acids, separate serum samples were used. Details can be found in attached method file.