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MB Sample ID: SA032651
| Local Sample ID: | Veh_01 |
| Subject ID: | SU000616 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammal |
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Sample Preparation:
| Sampleprep ID: | SP000623 |
| Sampleprep Summary: | Oxylipins, endocannabinoids, and fatty acids were isolated using a Waters Ostro Sample Preparation Plate (Milford, MA). Adipose samples were pulverized and aliquoted (~10-15mg) were added to 2mL polypropylene tubes and spiked with a 5 µL anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000nM analytical deuterated surrogates. A total of 50 µL of methanol was added and the tube was placed in a Geno/Grinder for 30 sec. An additional 550µL isopropanol w/ 10mM ammonium formate & 1% formic acid and 100 uL water was added and the tube was placed in a Geno/Grinder for 30 sec before being centrifuged at 10,000g for 5 min at room temp. The supernate was then transferred into the plate wells and samples were eluted into glass inserts containing 10 μL 20% glycerol by applying a vacuum at 15 Hg for 10 min. Eluent was dried by speed vacuum for 35 min at the medium BP setting, before switching to an aqueous setting for an additional 35 min. Once dry, samples were re-constituted with the internal standard 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-Phenyl 3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN), vortexed 1 min, transferred to a spin filter (0.1 µm, Millipore, Billerica, MA), centrifuged for 3 min at 6ºC at <4500g (rcf), before being transferred to 2 mL LC-MS amber vials. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal standard was used to quantify the recovery of surrogate standards. |
| Sampleprep Protocol Filename: | HQL-79_Lipid_Mediator_Data_Report.docx |
