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.