Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA338675

Local Sample ID:	RU322_MP
Subject ID:	SU003241
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP003248
Sampleprep Summary:	The MTBE extraction protocol, with laboratory modifications, was used to extract lipids and polar metabolites from the serum. The 10 μL internal standards (IS) mixture contains 15:0-18:1-d7-PC (2 ppm), 15:0-18:1-d7-PG (2 ppm), and L-tryptophan-(indole-d5) (10 ppm) were spiked into an aliquot of 50 μL serum. Then the sample was extracted by adding 600 μL MTBE and 150 μL MeOH and vortexed for 30 min at room temperature. Next, the sample was added with 200 μL water and centrifuged for 3 min at 13,697 g for phase separation. The upper portion containing serum lipids was transferred to another tube. The extraction was repeated by adding 100 μL water, 100 μL MeOH, and 300 μL MTBE. The sample was vortexed for an additional 10 min and centrifuged for 3 min at 13,697 g. The upper portion was mixed with the lower portion, and the combined solution was dried in a vacuum concentrator (Vacufuge plus Vacuum Concentrator, Eppendorf) for 3 h. The sample reconstitution was performed by adding 100 μL of reconstituted solution (ACN/IPA/water, v/v/v = 65/30/5). For the lower portion, 150 μL cold MeOH was added and stored under a -20°C environment for 2 h, followed by 10 min of 21,401 g centrifugation for protein precipitation. Next, the supernatant was dried using a vacuum concentrator overnight and then reconstituted by adding 100 μL reconstituted solution (ACN/water, v/v = 50/50). The protein precipitation was repeated by mixing 60 μL reconstituted sample with 120 μL cold ACN and then putting the mixtures in a -20°C freezer for an hour. After a 15 min centrifugation at 21,401 g under 4°C, super supernatants (120 μL) were collected and stored under -80°C before further analysis.

Sampleprep ID:

SP003248

Sampleprep Summary:

The MTBE extraction protocol, with laboratory modifications, was used to extract lipids and polar metabolites from the serum. The 10 μL internal standards (IS) mixture contains 15:0-18:1-d7-PC (2 ppm), 15:0-18:1-d7-PG (2 ppm), and L-tryptophan-(indole-d5) (10 ppm) were spiked into an aliquot of 50 μL serum. Then the sample was extracted by adding 600 μL MTBE and 150 μL MeOH and vortexed for 30 min at room temperature. Next, the sample was added with 200 μL water and centrifuged for 3 min at 13,697 g for phase separation. The upper portion containing serum lipids was transferred to another tube. The extraction was repeated by adding 100 μL water, 100 μL MeOH, and 300 μL MTBE. The sample was vortexed for an additional 10 min and centrifuged for 3 min at 13,697 g. The upper portion was mixed with the lower portion, and the combined solution was dried in a vacuum concentrator (Vacufuge plus Vacuum Concentrator, Eppendorf) for 3 h. The sample reconstitution was performed by adding 100 μL of reconstituted solution (ACN/IPA/water, v/v/v = 65/30/5). For the lower portion, 150 μL cold MeOH was added and stored under a -20°C environment for 2 h, followed by 10 min of 21,401 g centrifugation for protein precipitation. Next, the supernatant was dried using a vacuum concentrator overnight and then reconstituted by adding 100 μL reconstituted solution (ACN/water, v/v = 50/50). The protein precipitation was repeated by mixing 60 μL reconstituted sample with 120 μL cold ACN and then putting the mixtures in a -20°C freezer for an hour. After a 15 min centrifugation at 21,401 g under 4°C, super supernatants (120 μL) were collected and stored under -80°C before further analysis.