Bindley Bioscience Center

Sample Preparation

Sample Extraction Protocol - Lipids and/or Phospholipids:

**Note: Test these methods with a standard before performing on a real sample. Standard(s) should be spiked into a representative sample matrix (i.e., leaf tissue or cell cultures) to evaluate the recovery of desired compounds.

  1. Extraction solvent = chloroform/methanol/water (70/20/10, v/v/v)(may need to add BHT at 10 mg/L or more)
  2. Add 2-5x volume extraction solvent to tissue/cells depending on estimate of lipid concentration
  3. Vortex vigorously for 5 minutes at room temperature
  4. If possible, centrifuge at 4°C at 1500g or more for 5 minutes, OR allow to settle for 30 minutes at 4°C
  5. Remove bottom layer and transfer to a fresh epi tube
  6. To top layer, add a second volume of extraction solvent
  7. Vortex and centrifuge again
  8. Collect bottom layer and add to first bottom layer
  9. Speedvac until sample is completely dry OR store at -20°C
  10. Resuspend in 50 uL of initial mobile phase composition
  11. Submit for HPLC chromatography

Sample Extraction Protocol - Sugars (Monosaccharides):

**Note: Test these methods with a standard before performing on a real sample. Standard(s) should be spiked into a representative sample matrix (i.e., leaf tissue or cell cultures) to evaluate the recovery of desired compounds.

Tissue Extraction:

  1. Add 200 uL serum to an epi tube
  2. Add 400 uL MeOH. Remain at room temperature for 1 hour, vortex every 20 minutes.
  3. Place in -20 °C freezer for 30 minutes.
  4. Remove from freezer. Spin down while cold, 14,000 RPM for 10 minutes. {proteins will pellet}
  5. Remove the MeOH supernatant.
  6. Add 200 uL of water. Vortex.
  7. Spin down (13,000 g) for 10 minutes.
  8. Remove the MeOH/water supernatant.
  9. SpeedVac to dryness. Can store -80 °C.

MSTFA Sample Derivatization:

  1. Add 50 uL anhydrous pyridine via syringe.
  2. Remove 20 uL and add to a fresh epi. Freeze the other 30 uL aliquot as a back-up.
  3. To the 20 uL of anhydrous pyridine solution, add 10 uL methoxyamine HCl (50 mg/mL in pyridine).
  4. Heat at 60°C for 30 minutes.
  5. Add 20 uL of MSTFA.
  6. Heat at 60°C for 30 minutes.
  7. Transfer contents to a glass GC micro autosampler vial.
  8. Submit for GC/MS analysis.

Contact

Address
Purdue University
Metabolite Profiling Facility (MPF)
Bindley Bioscience Center
1203 W. State St.
West Lafayette, IN 47907-2010

Bruce Cooper, Ph.D.

Bruce Cooper, Ph.D.
Facility Director

 

Amber Hopf Janasch

Amber Hopf Jannasch
Laboratory Manager