Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA090803

Local Sample ID:	604
Subject ID:	SU001313
Subject Type:	Mammal
Subject Species:	Bos taurus
Taxonomy ID:	9913
Gender:	Female

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Sample Preparation:

Sampleprep ID:	SP001321
Sampleprep Summary:	Oxylipins and endocannabinoids were isolated using a Waters Ostro™ Sample Preparation Plate. Luteal samples were homogenized and 40 ± 8 mg were added to 2 mL polypropylene tubes spiked with a 5 µL antioxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000 nM analytical deuterated surrogates as previously described (Agrawal et al., 2017; La Frano et al., 2017). Samples were then mixed with 35 µL methanol, 550 µL isopropanol w/ 10 mM ammonium formate, 1% formic acid and 100 µL water, and the tube was placed in a Geno/Grinder 2010 (SPEX SamplePrep) for 30 sec and centrifuged at 10,000 x g for 5 min at room temperature. Supernatants were transferred into the Ostro plate wells and captured in glass inserts containing 10 μL of 20% glycerol in methanol by applying 15 mmHg of vacuum for 10 min. The eluent was dried under vacuum and reconstituted with 100 µL, 1:1 MeOH/ACN (v/v) containing 100 nM of 1-cyclohexyl ureido, 3 dodecanoic acid and 1-phenyl ureido, 3-hexanoic acid urea used as internal standards (gifts from Dr. B.D. Hammock, University of California, Davis). The samples were then vortexed and filtered at 0.1µm through PVDF membranes (Millipore) by centrifugation < 4500 x g (rcf) for 3 min at 6 ºC. The filtrate was transferred to inserts in amber glass and stored at -20 ºC for less than 48 hours before analysis by UPLC-MS/MS. Analytes in 5 μL extract aliquot were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH column (Waters) using published protocols for oxylipins and endocannabinoids (Agrawal et al., 2017; Pedersen and Newman, 2018). Samples were held at 10ºC. Separated residues were detected by negative mode electrospray ionization for oxylipins and positive mode electrospray ionization for endocannabinoids using multiple reaction monitoring on an API 6500 QTRAP (AB Sciex). Analytes were quantified using internal standard methods and 5- to 7-point calibration curves (r2 ≥ 0.997). Calibrants and internal standards were either synthesized [10,11-DHN, 10,11-DHHep, 10(11)-EpHep] or purchased from Cayman Chemical, Avanti Polar Lipids Inc., or Larodan Fine Lipids. Data was processed with AB Sciex MultiQuant version 3.0.2. The internal standards were used to quantify recovery of surrogate standards

Sampleprep ID:

SP001321

Sampleprep Summary:

Oxylipins and endocannabinoids were isolated using a Waters Ostro™ Sample Preparation Plate. Luteal samples were homogenized and 40 ± 8 mg were added to 2 mL polypropylene tubes spiked with a 5 µL antioxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000 nM analytical deuterated surrogates as previously described (Agrawal et al., 2017; La Frano et al., 2017). Samples were then mixed with 35 µL methanol, 550 µL isopropanol w/ 10 mM ammonium formate, 1% formic acid and 100 µL water, and the tube was placed in a Geno/Grinder 2010 (SPEX SamplePrep) for 30 sec and centrifuged at 10,000 x g for 5 min at room temperature. Supernatants were transferred into the Ostro plate wells and captured in glass inserts containing 10 μL of 20% glycerol in methanol by applying 15 mmHg of vacuum for 10 min. The eluent was dried under vacuum and reconstituted with 100 µL, 1:1 MeOH/ACN (v/v) containing 100 nM of 1-cyclohexyl ureido, 3 dodecanoic acid and 1-phenyl ureido, 3-hexanoic acid urea used as internal standards (gifts from Dr. B.D. Hammock, University of California, Davis). The samples were then vortexed and filtered at 0.1µm through PVDF membranes (Millipore) by centrifugation < 4500 x g (rcf) for 3 min at 6 ºC. The filtrate was transferred to inserts in amber glass and stored at -20 ºC for less than 48 hours before analysis by UPLC-MS/MS. Analytes in 5 μL extract aliquot were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH column (Waters) using published protocols for oxylipins and endocannabinoids (Agrawal et al., 2017; Pedersen and Newman, 2018). Samples were held at 10ºC. Separated residues were detected by negative mode electrospray ionization for oxylipins and positive mode electrospray ionization for endocannabinoids using multiple reaction monitoring on an API 6500 QTRAP (AB Sciex). Analytes were quantified using internal standard methods and 5- to 7-point calibration curves (r2 ≥ 0.997). Calibrants and internal standards were either synthesized [10,11-DHN, 10,11-DHHep, 10(11)-EpHep] or purchased from Cayman Chemical, Avanti Polar Lipids Inc., or Larodan Fine Lipids. Data was processed with AB Sciex MultiQuant version 3.0.2. The internal standards were used to quantify recovery of surrogate standards