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MB Sample ID: SA053083

Local Sample ID:q3June2014_2b
Subject ID:SU000946
Subject Type:Plasma samples
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:Pediatric samples
Human Trial Type:Observational
Species Group:Human

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Subject:

Subject ID:SU000946
Subject Type:Plasma samples
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:Pediatric samples
Human Trial Type:Observational
Species Group:Human

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
q3June2014_2bSA053083FL010034Quality ControlHRM_Sample_Type

Collection:

Collection ID:CO000940
Collection Summary:Please contact project PI, Frank D. Gilliland (gillilan@usc.edu), for sample collection details.
Sample Type:Sodium Heparin Plasma
Blood Serum Or Plasma:Plasma

Treatment:

Treatment ID:TR000960
Treatment Summary:Samples were received frozen in aliquouts of <250uL. Freeze-thaw history for study samples prior to receipt by the Emory URR is provided in the Study Design section. Prior to analysis, samples were thawed and prepared for HRM analysis using the standard protocols described in the Sample Preparation section.

Sample Preparation:

Sampleprep ID:SP000953
Sampleprep Summary:Samples were prepared for metabolomics analysis using established methods (Johnson et al. (2010). Analyst; Go et al. (2015). Tox Sci). Prior to analysis, urine aliquots were removed from storage at -80°C and thawed on ice. Each cryotube was then vortexed briefly to ensure homogeneity, and 50 μL was transferred to a clean microfuge tube. Immediately after, the urine was treated with 100 μL of ice-cold LC-MS grade acetonitrile (Sigma Aldrich) containing 2.5 μL of internal standard solution with eight stable isotopic chemicals selected to cover a range of chemical properties. Following addition of acetonitrile, urine was equilibrated for 30 min on ice, upon which precipitated proteins were removed by centrifuge (16.1 ×g at 4°C for 10 min). The resulting supernatant (100 μL) was removed, added to a low volume autosampler vial and maintained at 4°C until analysis (<22 h).
Sampleprep Protocol ID:HRM_SP_082016_01
Sampleprep Protocol Filename:EmoryUniversity_HRM_SP_082016_01.pdf
Sampleprep Protocol Comments:Date effective: 30 July 2016
Extraction Method:2:1 acetonitrile: sample followed by vortexing and centrifugation
Sample Spiking:2.5 uL [13C6]-D-glucose, [15N,13C5]-L-methionine, [13C5]-L-glutamic acid, [15N]-L-tyrosine, [3,3-13C2]-cystine, [trimethyl-13C3]-caffeine, [U-13C5, U-15N2]-L-glutamine, [15N]-indole

Combined analysis:

Analysis ID AN001476 AN001477
Analysis type MS MS
Chromatography type HILIC Reversed phase
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column Waters XBridge Amide (50 x 2.1mm,2.5um) Thermo Higgins C18 (50 x 2.1mm,3um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE
Units Peak intensity Peak intensity

Chromatography:

Chromatography ID:CH001035
Chromatography Summary:The HILIC column is operated parallel to reverse phase column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6-port switching valves. During operation of HILIC separation method, the MS is operated in positive ion mode and 10 μL of sample is injected onto the HILIC column while the reverse phase column is flushing with wash solution. Flow rate is maintained at 0.35 mL/min until 1.5 min, increased to 0.4 mL/min at 4 min and held for 1 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 2% formic acid (v/v) in LC-MS grade water. Initial mobile phase conditions are 22.5% A, 75% B, 2.5% C hold for 1.5 min, with linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min, resulting in a total analytical run time of 5 min. During the flushing phase (reverse phase analytical separation), the HILIC column is equilibrated with a wash solution of 77.5% A, 20% B, 2.5% C.
Methods ID:2% formic acid in LC-MS grade water
Methods Filename:20160920_posHILIC120kres5min_ESI_c18negwash.meth
Chromatography Comments:Triplicate injections for each chromatography mode
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters XBridge Amide (50 x 2.1mm,2.5um)
Column Temperature:60C
Flow Gradient:A= water, B= acetontrile, C= 2% formic acid in water; 22.5% A, 75% B, 2.5% C hold for 1.5 min, linear gradient to 77.5% A, 20% B, 2.5% C at 4 min, hold for 1 min
Flow Rate:0.35 mL/min for 1.5 min; linear increase to 0.4 mL/min at 4 min, hold for 1 min
Sample Injection:10 uL
Solvent A:100% water
Solvent B:100% acetonitrile
Analytical Time:5 min
Sample Loop Size:15 uL
Sample Syringe Size:100 uL
Chromatography Type:HILIC
  
Chromatography ID:CH001036
Chromatography Summary:The C18 column is operated parallel to the HILIC column for simultaneous analytical separation and column flushing through the use of a dual head HPLC pump equipped with 10-port and 6-port switching valves. During operation of the C18 method, the MS is operated in negative ion mode and 10 μL of sample is injected onto the C18 column while the HILIC column is flushing with wash solution. Flow rate is maintained at 0.4 mL/min until 1.5 min, increased to 0.5 mL/min at 2 min and held for 3 min. Solvent A is 100% LC-MS grade water, solvent B is 100% LC-MS grade acetonitrile and solvent C is 10mM ammonium acetate in LC-MS grade water. Initial mobile phase conditions are 60% A, 35% B, 5% C hold for 0.5 min, with linear gradient to 0% A, 95% B, 5% C at 1.5 min, hold for 3.5 min, resulting in a total analytical run time of 5 min. During the flushing phase (HILIC analytical separation), the C18 column is equilibrated with a wash solution of 0% A, 95% B, 5% C until 2.5 min, followed by an equilibration solution of 60% A, 35% B, 5% C for 2.5 min.
Methods ID:10mM ammonium acetate in LC-MS grade water
Methods Filename:20160920_negC18120kres5min_ESI_HILICposwash.meth
Chromatography Comments:Triplicate injections for each chromatography mode
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Thermo Higgins C18 (50 x 2.1mm,3um)
Column Temperature:60C
Flow Gradient:A= water, B= acetontrile, C= 10mM ammonium acetate in water; 60% A, 35% B, 5% C hold for 0.5 min, linear gradient to 0% A, 95% B, 5% C at 1.5 min, hold for 3 min
Flow Rate:0.4 mL/min for 1.5 min; linear increase to 0.5 mL/min at 2 min held for 3 min
Sample Injection:10 uL
Solvent A:100% water
Solvent B:100% acetonitrile
Analytical Time:5 min
Sample Loop Size:15 uL
Sample Syringe Size:100 uL
Chromatography Type:Reversed phase

MS:

MS ID:MS001360
Analysis ID:AN001476
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
Capillary Temperature:250C
Collision Gas:N2
Dry Gas Flow:45
Dry Gas Temp:150C
Mass Accuracy:< 3ppm
Spray Voltage:+3500
Activation Parameter:5e5
Activation Time:118ms
Interface Voltage:S-Lens RF level= 55
Resolution Setting:120,000
Scanning Range:85-1275
Analysis Protocol File:EmoryUniversity_HRM_QEHF-MS_092017_v1.pdf
  
MS ID:MS001361
Analysis ID:AN001477
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:NEGATIVE
Capillary Temperature:250C
Collision Gas:N2
Dry Gas Flow:45
Dry Gas Temp:150C
Mass Accuracy:< 3ppm
Spray Voltage:-4000
Activation Parameter:5e5
Activation Time:118ms
Interface Voltage:S-Lens RF level= 55
Resolution Setting:120,000
Scanning Range:85-1275
Analysis Protocol File:EmoryUniversity_HRM_QEHF-MS_092017_v1.pdf
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