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MB Sample ID: SA184044
Local Sample ID: | Day14-3 |
Subject ID: | SU002029 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Gender: | Male |
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Sample Preparation:
Sampleprep ID: | SP002035 |
Sampleprep Summary: | Lipids of tissues were extracted by the method of Bligh and Dyer. The extracted solutions were dried up with centrifugal evaporator, dissolved in methanol : isopropanol=1:1, and stored at −20°C. Fatty acid metabolites were further purified from tissues by solid-phase extraction using InertSep NH2 columns (GL Science) with deuterium-labelled internal standard (11(12)-EET-d11). Briefly, InertSep NH2 columns were precon- ditioned with 6mL of hexane and lipids extracted from tissues by the method of Bligh and Dyer were applied with 500μL of chloroform. Columns were then washed with 6mL of chloro- form/isopropanol (2/1, v/v), followed by the elution with diethyl ether/acetic acid (98/2, v/v).The extracted solutions were dried up with centrifugal evaporator, dissolved in methanol:isopro- panol=1:1, and stored at −20°C. For the detection of fatty acid metabolites, chromatographic separation was performed on a ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm; Waters) maintained at 40°C using mobile phase A (water/acetic acid (100/0.1, v/v) containing 10 mM ammonium acetate) and mobile phase B (acetonitrile/methanol (4/1, v/v) containing 10 mM ammonium acetate) in a gradient programme (0–2 min: 90% A; 2–10 min: 90% A →30% A; 10–24 min: 30% A →27% A; 24–27 min: 1% A; 27–32 min: 90% A) with a flow rate of 0.2 mL/min(0–10 min), 0.1 mL/min(10–15 min),0.2 mL/min(15–24 min) and 0.5 mL/min(24–32 min). |