Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA046788

Local Sample ID:	AZ 13
Subject ID:	SU000870
Subject Type:	Animal
Subject Species:	Macaca mulatta
Taxonomy ID:	9544
Species Group:	Mammal

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Subject:

Subject ID:	SU000870
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
AZ 13	SA046788	FL009238	Control	Treatment
AZ 13	SA046788	FL009238	Left Lung	Lung
AZ 13	SA046788	FL009238	12	Time point (day)

Collection:

Collection ID:	CO000864
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. Lung wash fluid was collected at day 0, 8 and 12 of each treatment.
Sample Type:	Bronchoalveolar lavage

Treatment:

Treatment ID:	TR000884
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. Lung wash fluid was collected at day 0, 8 and 12 of each treatment.

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.

Combined analysis:

Analysis ID	AN001364	AN001365
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity
Column	Waters Acquity BEH C18 (100 x 2mm,1.7um)	Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 6500 QTrap	ABI Sciex 6500 QTrap
Ion Mode	NEGATIVE	POSITIVE
Units	Concentration (nM)	Concentration (nM)

Chromatography:

Chromatography ID:	CH000952
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2mm,1.7um)
Column Temperature:	60 °C
Flow Gradient:	See protocol/methods file
Flow Rate:	0.25 mL/min
Internal Standard:	See protocol/methods file
Retention Time:	See protocol/methods file
Sample Injection:	5 µL
Solvent A:	100% water; 0.1% acetic acid
Solvent B:	90% acetonitrile/ 10% isopropanol
Analytical Time:	20 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:	MS001256
Analysis ID:	AN001364
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	NEGATIVE

MS ID:	MS001257
Analysis ID:	AN001365
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE