Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA037922

Local Sample ID:	P1 E10
Subject ID:	SU000682
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male
Subject Comments:	Individuals with peripheral arterial disease, as defined by ankle brachial indices ≥0.4 and ≤0.8 and the presence of classic claudication symptoms
Species Group:	Human

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Sample Preparation:

Sampleprep ID:	SP000689
Sampleprep Summary:	Lipid mediator extraction and quantification: Oxylipins, endocannabinoids, and fatty acids were isolated from plasma using Ostro Sample Preparation Plates (Waters; Milford, MA) in the presence of BHT/EDTA and quantified by UPLC-MS/MS using internal standard methods. Briefly, in the Ostro Plate well, 100µl plasma aliquots were mixed with 5µl of BHT/EDTA (1:1 v/v MeOH/water) and 5µl methanol containing a suite of deuterated surrogates. The samples were then mixed with 300µL of acetonitrile with 1% formic acid and eluted with 15 mmHg vacuum for 10 min into 200µL glass inserts containing 10µl of 20% glycerol in methanol. Solvent was removed by vacuum centrifugation and the glycerol plug was stored at -80Co until analysis. Prior acquisition, samples were reconstituted in 100µl of 1:1 methanol:acetonitrile containing 100nM of 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-phenyl ureido, 3-hexadecanoic acid (PUHA). Residues within extracts were separated on a 2.1 x 150mm 0.17µm BEH column (Waters) and detected by electrospray ionization with multi reaction monitoring on a API 6500 QTRAP (Sciex; Redwood City, CA) and quantified against 7-9 point calibration curves of authentic standards using modifications of previously reported methods (Grapov, D., et al. 2012. PLoS One 7: e48852.; doi: 10.1371/journal.pone.0048852).

Sampleprep ID:

SP000689

Sampleprep Summary:

Lipid mediator extraction and quantification: Oxylipins, endocannabinoids, and fatty acids were isolated from plasma using Ostro Sample Preparation Plates (Waters; Milford, MA) in the presence of BHT/EDTA and quantified by UPLC-MS/MS using internal standard methods. Briefly, in the Ostro Plate well, 100µl plasma aliquots were mixed with 5µl of BHT/EDTA (1:1 v/v MeOH/water) and 5µl methanol containing a suite of deuterated surrogates. The samples were then mixed with 300µL of acetonitrile with 1% formic acid and eluted with 15 mmHg vacuum for 10 min into 200µL glass inserts containing 10µl of 20% glycerol in methanol. Solvent was removed by vacuum centrifugation and the glycerol plug was stored at -80Co until analysis. Prior acquisition, samples were reconstituted in 100µl of 1:1 methanol:acetonitrile containing 100nM of 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-phenyl ureido, 3-hexadecanoic acid (PUHA). Residues within extracts were separated on a 2.1 x 150mm 0.17µm BEH column (Waters) and detected by electrospray ionization with multi reaction monitoring on a API 6500 QTRAP (Sciex; Redwood City, CA) and quantified against 7-9 point calibration curves of authentic standards using modifications of previously reported methods (Grapov, D., et al. 2012. PLoS One 7: e48852.; doi: 10.1371/journal.pone.0048852).