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MB Sample ID: SA220285

Local Sample ID:NP_B
Subject ID:SU002378
Subject Type:Water sample

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Subject:

Subject ID:SU002378
Subject Type:Water sample

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
NP_BSA220285FL027458CX-SPEExtraction_Approach
NP_BSA220285FL027458North PacificLocation

Collection:

Collection ID:CO002371
Collection Summary:Samples for dissolved metabolites from four unique environmental locations (Lake Washington (LW), Puget Sound (PS), Station ALOHA in the North Pacific Subtropical Gyre (ALOHA), and the North Pacific (NP)) from depths of 15 m for ALOHA and NP samples, 8 m for the PS sample, and 1 m for the LW sample. Samples were processed by filtering the water through 147 mm 0.2 μm PTFE filters using peristaltic pumps (ALOHA, NP, PS samples) and or glass vacuum filtration onto a 47 mm 0.2 μm PTFE filter. The filtrated was collected and stored in 2 L acid-washed polycarbonate bottles (ALOHA, NP, PS samples) or 45 mL acid-washed polypropylene falcon tubes at -20 C for between 1 month and 4 years until analysis.
Sample Type:seawater
Storage Conditions:-20℃

Treatment:

Treatment ID:TR002390
Treatment Summary:Not Applicable

Sample Preparation:

Sampleprep ID:SP002384
Sampleprep Summary:Cation-Exchange Solid Phase Extraction (CX-SPE) Sample Volume: 40 mLs Solid Phase Resin/Column: Strong cation-exchange resin (Dowex 50WX8; H+ form, 100-200 mesh, Sigma-Aldrich, Vienna, Austria) Solvents/Reagents: 1 M NH3, 3 M HNO3 Brief Procedure: 35 g of resin was added to a glass chromatography column with a fritted disk and a PTFE stopcock. The resin was equilibrated with 50 mL water, 100 mL of 1M NH3, 50 mL water, 100 mL 3M HNO3, and 50 mL H2O. The samples were acidified with HNO3 to pH 2 and heavy isotope-labeled internal standards added. The sample was loaded onto column, allowed to stand for 5 minutes, and then drained from the column. The column was then rinsed with 50 mL water. Approximately 200 mL of 1M NH3 was added to column. Ammonia eluted from column in 10 mL fractions. The pH of each fraction checked by dabbing a small drop of sample onto a pH strip with a combusted glass Pasteur pipette. The alkaline front (the 10 mL fraction where the pH increases from approximately 2-4 to 9-11), the fraction before, and the two fractions after were collected, combined, and dried down under N2 gas in a water bath of approximately 32 degrees C. Dried fractions were redissolved in 380 uL of water. 20 uL of isotope-labeled injection standards in water were added to the fractions. Columns were regenerated for reuse through the addition of 50 mL of water, 100 mL 3M HNO3, and 50 mL of water. When not in use, columns were stored completely filled with 0.01 M HNO3. Water was extracted alongside samples as methodological blanks. PPL-Solid Phase Extraction (PPL-SPE) Sample Volume: 40 mLs Solid Phase Resin/Column: Agilent Bond Elut PPL cartridge, 1 g bed mass, 6 mL volume Solvents/Reagents: methanol, 0.01 M HCl, Brief Procedure: Sample acidified with HCl to pH 2 and heavy isotope-labeled internal standards added. Column prepped by adding 1 cartridge volumes of each of methanol followed by 0.01 M HCl. Sample loaded onto columns using a peristaltic pump at a flow rate of 10 mL/min Column rinsed with 2 cartridge volumes 0.01 M HCl. Sample eluted with 1 cartridge volume methanol and dried down under N2 gas. Dried fractions were redissolved in 380 uL of water. 20 uL of isotope-labeled injection standards in water were added to the fractions. Water was extracted alongside samples as methodological blanks.

Combined analysis:

Analysis ID AN003744 AN003745 AN003746
Analysis type MS MS MS
Chromatography type HILIC HILIC Reversed phase
Chromatography system Waters Acquity I-Class Waters Acquity I-Class Waters Acquity I-Class
Column SeQuant ZIC-pHILIC (150 x 2.1mm,5um) SeQuant ZIC-pHILIC (150 x 2.1mm,5um) Waters Acquity HSS Cyano (1.8um,2.1 mm x 100 mm)
MS Type ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE POSITIVE
Units Peak Area Peak Area Peak Area

Chromatography:

Chromatography ID:CH002774
Chromatography Summary:See attached summary
Methods Filename:Ingalls_Lab_LC_Methods_2.txt
Instrument Name:Waters Acquity I-Class
Column Name:SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:30
Flow Rate:0.15 ml/min
Solvent A:85% acetonitrile/15% water; 10 mM ammonium carbonate
Solvent B:15% acetonitrile/85% water; 10 mM ammonium carbonate
Chromatography Type:HILIC
  
Chromatography ID:CH002775
Chromatography Summary:See attached summary
Methods Filename:Ingalls_Lab_LC_Methods_2.txt
Instrument Name:Waters Acquity I-Class
Column Name:Waters Acquity HSS Cyano (1.8um,2.1 mm x 100 mm)
Column Temperature:35
Flow Rate:0.4 ml/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003491
Analysis ID:AN003744
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:See Protocol
Ion Mode:POSITIVE
Analysis Protocol File:CXSPE_Method_Paper_MS_Methods.txt
  
MS ID:MS003492
Analysis ID:AN003745
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:See Protocol
Ion Mode:NEGATIVE
Analysis Protocol File:CXSPE_Method_Paper_MS_Methods.txt
  
MS ID:MS003493
Analysis ID:AN003746
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:See Protocol
Ion Mode:POSITIVE
Analysis Protocol File:CXSPE_Method_Paper_MS_Methods.txt
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