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MB Sample ID: SA032765

Local Sample ID:LAM-05
Subject ID:SU000620
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Species Group:Mammal

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Sample Preparation:

Sampleprep ID:SP000627
Sampleprep Summary:Oxylipins and endocannabinoids were isolated using a Waters Ostro Sample Preparation Plate (Milford, MA). Hypothalamus samples were pulverized and aliquoted (~20-25mg) 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 100 µL methanol was added to the sample and vrtexed 90 sec. Next, 500 µL D.I. water and 1000 µL ethyl acetate was added and the tube was vortexed 3 minutes, before being centrifuged at 15,000g for 10 min at room temp. The supernate was then transferred into a clean 2 mL autosampler vial. The extraction with ethyl acetate was repeated and the eluent was dried by speed vacuum for 35 min at the medium BP setting. 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:Hypothal_Newman_Data_Report.docx