Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA046784

Local Sample ID:	AZ 16
Subject ID:	SU000870
Subject Type:	Animal
Subject Species:	Macaca mulatta
Taxonomy ID:	9544
Species Group:	Mammal

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Sample Preparation:

Sampleprep ID:	SP000877
Sampleprep Summary:	Oxylipins, endocannabinoids, and fatty acids were isolated by solid phase extraction on 60 mg Waters Oasis-HLB cartridges (Milford, MA), as previously described by Luria et al (1). Prior to extraction, cartridges were washed with 1 column volume ethyl acetate followed by 2 column volumes methanol and conditioned with 2 mL of 95:5 v/v water/methanol (MeOH) with 0.1% acetic acid. The column reservoir was spiked with 5 µL anti-oxidant solution, (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water), and 10 μL 1000nM analytical surrogates (See Table 2 below for specific compounds). Sample aliquots (500 µL media) were then introduced to the column reservoir and diluted with 1 column volume wash solution (5% MeOH, 0.1% acetic acid) and allowed to gravity extract into tubes containing 10% bleach solution to decontaminate sample waste. SPE cartridges were dried by vacuum @ -7.5in Hg for 20 min. Analytes were then eluted by gravity with 0.5 mL MeOH, followed by 1.25 mL Ethyl Acetate, into 2 mL autosampler vials containing 10 µL 20% glycerol solution in MeOH. Eluent was dried by vacuum evaporation for 35 min, and residues were re-constituted with 100uL of 100 nM internal standard solution containing 1-cyclohexyl ureido,3-dodecanoic acid (CUDA) and 1-Phenyl 3-Hexadecanoic Acid Urea (PHAU), in 50:50 MeOH:ACN and let sit for 10 min at ambient temperature. Vials were then chilled 15 min on wet ice, and extracts were transferred to a centrifugal filter (0.1 µm Durapore, Millipore, Billerica, MA), centrifuged for 3 min at 6ºC at <4500g (rcf) and transferred to 150 uL glass inserts within 2 mL amber vials, and capped. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal standard was used to quantify the recovery of the deuterated extraction surrogates by ratio response.

Sampleprep ID:

SP000877

Sampleprep Summary:

Oxylipins, endocannabinoids, and fatty acids were isolated by solid phase extraction on 60 mg Waters Oasis-HLB cartridges (Milford, MA), as previously described by Luria et al (1). Prior to extraction, cartridges were washed with 1 column volume ethyl acetate followed by 2 column volumes methanol and conditioned with 2 mL of 95:5 v/v water/methanol (MeOH) with 0.1% acetic acid. The column reservoir was spiked with 5 µL anti-oxidant solution, (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water), and 10 μL 1000nM analytical surrogates (See Table 2 below for specific compounds). Sample aliquots (500 µL media) were then introduced to the column reservoir and diluted with 1 column volume wash solution (5% MeOH, 0.1% acetic acid) and allowed to gravity extract into tubes containing 10% bleach solution to decontaminate sample waste. SPE cartridges were dried by vacuum @ -7.5in Hg for 20 min. Analytes were then eluted by gravity with 0.5 mL MeOH, followed by 1.25 mL Ethyl Acetate, into 2 mL autosampler vials containing 10 µL 20% glycerol solution in MeOH. Eluent was dried by vacuum evaporation for 35 min, and residues were re-constituted with 100uL of 100 nM internal standard solution containing 1-cyclohexyl ureido,3-dodecanoic acid (CUDA) and 1-Phenyl 3-Hexadecanoic Acid Urea (PHAU), in 50:50 MeOH:ACN and let sit for 10 min at ambient temperature. Vials were then chilled 15 min on wet ice, and extracts were transferred to a centrifugal filter (0.1 µm Durapore, Millipore, Billerica, MA), centrifuged for 3 min at 6ºC at <4500g (rcf) and transferred to 150 uL glass inserts within 2 mL amber vials, and capped. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal standard was used to quantify the recovery of the deuterated extraction surrogates by ratio response.