Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA331355

Local Sample ID:	Donor_5_Serum_1
Subject ID:	SU003165
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Sample Preparation:

Sampleprep ID:	SP003171
Sampleprep Summary:	SERUM/PLASMA: Frozen EDTA-anticoagulated plasma or serum was freshly thawed on ice. For precipitation of proteins, plasma or serum (400 µL) was mixed with cold EtOH (1.6 mL, abs. 99%, -20°C; AustroAlco) including an internal standard mixture of 12S-HETE-d8, 15S-HETE-d8, 5-Oxo-ETE-d7, 11,12-DiHETrE-d11, PGE2-d4 and 20-HETE-d6 (concentrations can be found below). The samples were stored over-night at -20°C. After centrifugation (30 min, 4536 g, 4°C), the supernatant was transferred into a new 15 mL FalconTM tube. EtOH was evaporated via vacuum centrifugation at 37°C until the original sample volume (400 µL) was restored. For solid phase extraction (SPE) samples were loaded onto preconditioned StrataX SPE columns (30 mg mL-1; Phenomenex, Torrance, CA, USA) using Pasteur pipettes. After sample loading, the SPE columns were washed with 5 mL of MS grade water and eluted with ice-cold MeOH (500 µL; MeOH abs.; VWR International, Vienna, Austria) containing 2% formic acid (FA; Sigma-Aldrich). MeOH was evaporated using a gentle nitrogen stream at room temperature and the dried samples were reconstituted in 150 µL reconstitution buffer (H2O:ACN:MeOH + 0.2% FA–vol% 65:31.5:3.5). The samples were then transferred into an autosampler held at stored at 4°C and subsequently measured via LC-MS/MS. 12S-HETE-d8: 6.67 pg/µL 15S-HETE-d8: 6.67 pg/µL 5-Oxo-ETE-d7: 20 pg/µL 11,12-DiHETrE-d11: 6.67 pg/µL PGE2-d4: 13.33 pg/µL 20-HETE-d6: 6.67 pg/µL

Sampleprep ID:

SP003171

Sampleprep Summary:

SERUM/PLASMA: Frozen EDTA-anticoagulated plasma or serum was freshly thawed on ice. For precipitation of proteins, plasma or serum (400 µL) was mixed with cold EtOH (1.6 mL, abs. 99%, -20°C; AustroAlco) including an internal standard mixture of 12S-HETE-d8, 15S-HETE-d8, 5-Oxo-ETE-d7, 11,12-DiHETrE-d11, PGE2-d4 and 20-HETE-d6 (concentrations can be found below). The samples were stored over-night at -20°C. After centrifugation (30 min, 4536 g, 4°C), the supernatant was transferred into a new 15 mL FalconTM tube. EtOH was evaporated via vacuum centrifugation at 37°C until the original sample volume (400 µL) was restored. For solid phase extraction (SPE) samples were loaded onto preconditioned StrataX SPE columns (30 mg mL-1; Phenomenex, Torrance, CA, USA) using Pasteur pipettes. After sample loading, the SPE columns were washed with 5 mL of MS grade water and eluted with ice-cold MeOH (500 µL; MeOH abs.; VWR International, Vienna, Austria) containing 2% formic acid (FA; Sigma-Aldrich). MeOH was evaporated using a gentle nitrogen stream at room temperature and the dried samples were reconstituted in 150 µL reconstitution buffer (H2O:ACN:MeOH + 0.2% FA–vol% 65:31.5:3.5). The samples were then transferred into an autosampler held at stored at 4°C and subsequently measured via LC-MS/MS. 12S-HETE-d8: 6.67 pg/µL 15S-HETE-d8: 6.67 pg/µL 5-Oxo-ETE-d7: 20 pg/µL 11,12-DiHETrE-d11: 6.67 pg/µL PGE2-d4: 13.33 pg/µL 20-HETE-d6: 6.67 pg/µL