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MB Sample ID: SA331361
Local Sample ID: | Donor_1_Serum_after_Aspirin_1 |
Subject ID: | SU003165 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003165 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Donor_1_Serum_after_Aspirin_1 | SA331361 | FL039184 | Serum | Sample material |
Donor_1_Serum_after_Aspirin_1 | SA331361 | FL039184 | after_Aspirin | Treatment |
Collection:
Collection ID: | CO003158 |
Collection Summary: | SERUM/PLASMA: Blood samples were obtained at baseline and after 7 days intake of the study medication. On both study days two blood samples using 6 mL K3EDTA and serum collection tubes (both Vacuette, Greiner Bio-One GmbH, Kremsmünster, Austria) were obtained from each subject. EDTA-anticoagulated tubes were carefully inverted two times after blood draw and centrifuged immediately at room temperature at 2000 g for 10 min. In contrast, filled serum tubes were carefully inverted after blood draw and placed to sit upright for 15 to 30 minutes to allow clot formation. Then, tubes were centrifuged at room temperature at 2000 g for 10 min. Directly after centrifugation, 500 µL of plasma or serum, respectively, were transferred into pre-labelled Eppendorf safe-lock tubes and stored at -80°C until analysis. |
Sample Type: | Blood (serum) and blood (plasma) |
Treatment:
Treatment ID: | TR003174 |
Treatment Summary: | Subjects were randomized to receive either acetylsalicylic acid or Omega-3 capsules for 7 days. One study cohort was instructed to take 500 mg acetylsalicylic acid (Aspirin® 500 mg acetylsalicylic acid, Cellulose powder, maize starche) per day in the evening whereas the second study cohort was instructed to take two Omega-3 complex 870 mg capsules (Br. Böhm Omega-3 capsules, 1017 mg cold water fish oil equivalent to 870 mg Omega-3, consisting of 420 mg EPA, 330 mg DHA, 5μg Vitamin D equivalent to 200 IU, 6 mg Vitamin E, 30 mg Co-enzyme Q10) per day in the evening. |
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 |
Combined analysis:
Analysis ID | AN005001 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Thermo Vanquish |
Column | Phenomenex Kinetex XB-C18 (150 x 2.1mm, 2.6um) |
MS Type | ESI |
MS instrument type | Orbitrap |
MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
Ion Mode | NEGATIVE |
Units | normalized AUC |
Chromatography:
Chromatography ID: | CH003779 |
Chromatography Summary: | For LC-MS analyses, analytes were separated using a Thermo Scientific Vanquish (UHPLC) system equipped with a Kinetex C18-column (2.6 µm, XB-C18, 100 A° , LC Column 150 * 2.1 mm; Phenomenex) applying a gradient flow profile (mobile phase A: H2O + 0.2% FA, mobile phase B: ACN:MeOH (vol% 90:10) + 0.2% FA) starting at 35% B and increasing to 90% B (1–10 min), further increasing to 99% B within 0.5 min and held for 5 min. Solvent B was then decreased to the initial level of 35% within 0.5 min and the column was equilibrated for 4 min, resulting in a total run time of 20 min. The flow rate was kept at 200 µL min-1 and the column oven temperature at 40°C. The injection volume was 20 µL and all samples were analysed in technical duplicates. |
Instrument Name: | Thermo Vanquish |
Column Name: | Phenomenex Kinetex XB-C18 (150 x 2.1mm, 2.6um) |
Column Temperature: | 40 |
Flow Gradient: | 0min with 35% B to 90% B (1–10 min), further increasing to 99% B within 0.5 min and held for 5 min. Solvent B was then decreased to the initial level of 35% within 0.5 min and the column was equilibrated for 4 min, resulting in a total run time of 20 min. |
Flow Rate: | 200 µL/min |
Solvent A: | 100% water; 0.2% formic acid |
Solvent B: | 90% acetonitrile/10% methanol; 0.2% formic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS004741 |
Analysis ID: | AN005001 |
Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | The Vanquish UHPLC system was coupled to a Q ExactiveTM HF Quadrupole-OrbitrapTM high-resolution mass spectrometer (Thermo Fisher Scientific, Austria), equipped with a HESI source for negative ionization to perform the mass spectrometric analysis. The MS scan range was 250-700 m/z with a resolution of 60,000 (at m/z 200) on the MS1 level. A Top 2 method was applied for fragmentation (HCD 24 normalized collision energy), preferable 33 m/z values specific for well-known eicosanoids and precursor molecules from an inclusion list. The resulting fragments were analysed on the MS2 level at a resolution of 15,000 (at m/z 200). Operating in negative ionization mode, a spray voltage of 3.5 kV and a capillary temperature of 253°C were applied. Sheath gas was set to 46 and the auxiliary gas to 10 (arbitrary units). |
Ion Mode: | NEGATIVE |