Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA046944

Local Sample ID:	Zeki L 40
Subject ID:	SU000874
Subject Type:	Animal
Subject Species:	Macaca mulatta
Taxonomy ID:	9544
Species Group:	Mammal

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Subject:

Subject ID:	SU000874
Subject Type:	Animal
Subject Species:	Macaca mulatta
Taxonomy ID:	9544
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Zeki L 40	SA046944	FL009280	Left Lung	Tissue
Zeki L 40	SA046944	FL009280	Simvastatin	Treatment

Collection:

Collection ID:	CO000868
Collection Summary:	Monkeys were treated with placebo or Provastatin for 12 days. Further, after the wash out period animals were treated with Simvastatin for 12 days. Plasma was collected at day 0, 8 and 12 of each treatment.
Sample Type:	Tissue
Tissue Cell Identification:	Trachea and lung tissue

Treatment:

Treatment ID:	TR000888
Treatment Summary:	Monkeys were treated (by inhalation) with placebo or Provastatin for 12 days. Further, after the wash out period animals were treated with Simvastatin for 12 days. Plasma was collected at day 0, 8 and 12 of each treatment.

Sample Preparation:

Sampleprep ID:	SP000881
Sampleprep Summary:	Oxylipins, endocannabinoids, bile acids and fatty acids were isolated using a Waters Ostro Sample Preparation Plate (Milford, MA). Lung and trachea samples were aliquoted (~40-60mg) into 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, 550µL isopropanol w/ 10mM ammonium formate & 1% formic acid and 100 uL water were 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 ID:

SP000881

Sampleprep Summary:

Oxylipins, endocannabinoids, bile acids and fatty acids were isolated using a Waters Ostro Sample Preparation Plate (Milford, MA). Lung and trachea samples were aliquoted (~40-60mg) into 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, 550µL isopropanol w/ 10mM ammonium formate & 1% formic acid and 100 uL water were 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.

Combined analysis:

Analysis ID	AN001371
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Aquity C18 BEH (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex 6500 QTrap
Ion Mode	NEGATIVE
Units	Concentration (nM)

Chromatography:

Chromatography ID:	CH000956
Instrument Name:	Waters Acquity
Column Name:	Aquity C18 BEH (100 x 2.1mm,1.7um)
Column Temperature:	60 °C
Flow Gradient:	See protocol/methods file
Flow Rate:	0.4 mL/min
Internal Standard:	See protocol/methods file
Retention Time:	See protocol/methods file
Sample Injection:	5 µL
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Analytical Time:	16 min
Weak Wash Solvent Name:	20% methanol, 10% isopropanol
Weak Wash Volume:	600 µL
Strong Wash Solvent Name:	50:50 Acetonitrile:Methanol
Strong Wash Volume:	600 µL
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001263
Analysis ID:	AN001371
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	NEGATIVE