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MB Sample ID: SA091431

Local Sample ID:Sample067
Subject ID:SU001327
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Male and female

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Sample Preparation:

Sampleprep ID:SP001335
Sampleprep Summary:All samples were removed from -80°C and allowed to thaw on ice. Of the 100-250 uL of plasma shipped to the facility for analysis, 30 µL of plasma was pipetted into a new 1.5 mL Eppendorf tube. 1 mL of 3:3:2 acetonitrile:isopropanol:water was added to each sample. Samples were vortexed for 10 seconds using a MiniVortexer. Samples were then shaken on an Orbital Mixing Chilling/Heating Plate for 5 minutes at 4°C. Samples were centrifuged for 2 minutes at 14,000 rcf used the centrifuge Eppendorf 5415 D. Two 475 µL aliquots were removed from the supernatant. Both samples were dried to complete using Labconco Centrivap cold trap. 500 µL of 50:50 acetonitrile was added to each aliquot of all samples. Samples were vortexed for 10 seconds using the MiniVortexer. Samples were centrifuged for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415 D. 475 µL of supernatant was removed and evaporated to dryness using the Labconco Centrivap cold trap. One sample is submitted to derivitization, the other sample is saved for LC-MS analysis.
Extraction Method:Extraction is carried out using a bi-phasic solvent system of cold methanol, methyl tert-butyl ether (MTBE), and water. In more detail, cold methanol (225 µL is added to a 5mg tissue sample aliquot, which is placed into a 1.5 mL Eppendorf tube. Then, 750 µL of cold MTBE is added, followed by vortexing for 10 s. and shaking for 6 min. at 4ºC. Phase separation is induced by adding 188 µL of mass spec-grade water. After vortexing for 20 s. the sample is centrifuged at 14,000 rpm for 2 min. The upper organic phase is collected in two 300 µL aliquots for lipid analysis polar layer is collected in two 125 µL aliquots for HILIC analysis. One is stored at -20ºC as a backup and the other is evaporated to dryness in a SpeedVac. Dried extracts are resuspended in acetonitrile.
Sample Derivatization:None
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