Extraction of Lipid from Tissue Sample by MTBE

1. Reagent

   1. Ammonium Acetate

   2. Methanol

   2. Methoyl Tertiary Butyl Ether(MTBE)

2. Material 

   1. EP tube

Step 1

• Tissue
  Mince tissue into small pieces in 0.1% ammonium acetate water solution followed by homogenization.

• Serum/Plasma

Step 2

1. 100 μL Step 1 sample aliquot add 750 μL methanol and vortex for at least 10s in the EP tube.

2. Add 2.5 mL MTBE and mix well by vortex or ultrasonic 

3. Incubate the mixture at RT for 1 h in a shaker.

Step 3

1. Add 625 μL H2O and mix well.

2. Stay at RT for 10 min.

3. Centrifuge at 1000 g for 10 min.

Step 4

1. Take the supernate (organic phase) to another EP tube.

2. Re-extract the lower phase  with 2 mL of extraction agent (will it be incubate as Step 2?).

3. Combine the 4.2 supernate with 4.1.

Step 5

1. Concentrate the combined supernate with nitrogen or vacuum centrifuge.

2. Redissolve the 5.1 by 100 μL mobile phase A (ACN:H2O, 60%:40%) for injection.