Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA318777

Local Sample ID:	CB086-V
Subject ID:	SU003050
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP003056
Sampleprep Summary:	Metabolites and lipids were extracted in 96-well high throughput fashion using a liquid-liquid biphasic separation with cold methyl tert-butyl ether (MTBE), methanol, and water. To begin, 1 mL MTBE was added to 40 μl of plasma and spiked with 40 μl of deuterated lipid internal standards (Sciex, cat# 5040156, lot# LPISTDKIT-103). The samples were agitated at 4°C for 30 minutes. After the addition of 250 μl cold water, samples were vortexed for 1 minute then centrifuged at 3,800 g for 5 minutes at 4°C. The upper organic phase contained the lipids while the lower aqueous phase contained metabolites with precipitated proteins at the bottom of the tube. For quality control, reference plasma samples (40 μl plasma), as well as controls lacking samples (blanks), were processed in parallel. 1) Metabolites: To further precipitate proteins, 500 μl 1:1:1 acetone: acetonitrile: methanol spiked with 16 labeled metabolite internal standards was added to 300 μl of the aqueous phase and 200 μl of the organic phase and incubated overnight at -20°C. After centrifugation at 3,800 g for 10 min at 4°C, the metabolic extracts were dried down under a stream of nitrogen gas and resuspended in 100 μl 50/50 methanol/water for LC-MS. 2) Complex lipids: 700 µl of the organic phase was dried down under a stream of nitrogen and resolubilized in 200 μl of methanol for storage at -20°C until analysis. The day of the analysis, samples were dried down, resuspended in 300 μl of 10 mM ammonium acetate in 90/10 methanol/toluene, and centrifuged at 3,800 g for 5 min at 4°C.

Sampleprep ID:

SP003056

Sampleprep Summary:

Metabolites and lipids were extracted in 96-well high throughput fashion using a liquid-liquid biphasic separation with cold methyl tert-butyl ether (MTBE), methanol, and water. To begin, 1 mL MTBE was added to 40 μl of plasma and spiked with 40 μl of deuterated lipid internal standards (Sciex, cat# 5040156, lot# LPISTDKIT-103). The samples were agitated at 4°C for 30 minutes. After the addition of 250 μl cold water, samples were vortexed for 1 minute then centrifuged at 3,800 g for 5 minutes at 4°C. The upper organic phase contained the lipids while the lower aqueous phase contained metabolites with precipitated proteins at the bottom of the tube. For quality control, reference plasma samples (40 μl plasma), as well as controls lacking samples (blanks), were processed in parallel. 1) Metabolites: To further precipitate proteins, 500 μl 1:1:1 acetone: acetonitrile: methanol spiked with 16 labeled metabolite internal standards was added to 300 μl of the aqueous phase and 200 μl of the organic phase and incubated overnight at -20°C. After centrifugation at 3,800 g for 10 min at 4°C, the metabolic extracts were dried down under a stream of nitrogen gas and resuspended in 100 μl 50/50 methanol/water for LC-MS. 2) Complex lipids: 700 µl of the organic phase was dried down under a stream of nitrogen and resolubilized in 200 μl of methanol for storage at -20°C until analysis. The day of the analysis, samples were dried down, resuspended in 300 μl of 10 mM ammonium acetate in 90/10 methanol/toluene, and centrifuged at 3,800 g for 5 min at 4°C.