The total volume of lipid extract for Lipid analysis is 4 ml.
From this extract 0.1ml aliquot is taken for SM base hydrolysis and dried down under Nitrogen.
To the dried extract 1 ml Methanol is added and then 20 ul 1N NaON. The sodium hydroxide solution is prepared in methanol.
The final concentration of NaOH in the sample now is ≈0.02N (0.0196N) Vortex the sample.
The samples are left to hydrolyze for 3 hour at room temperature.
After 3 hours the samples are vortexed again.
To extract the SM -
- 1ml of Chloroform is added and the sample vortexed again.
- After that 1 ml of Methanol and vortexed.
- Then 0.8 ml 1N NaCl and vortex. The concentration of NaCl is 1N in water.
- Then 1 ml Chloroform
- Then 1 ml 1N NaCl (aq)
- Then vortex for a longer period of time to extract the lipids.
Samples are centrifuged for 5 min at 3000 rpm.
The upper aqueous layer is taken out and discarded.
The bottom organic layer containing the lipids is dried down under Nitrogen.
The final sample is prepared by re-suspending the dried down lipids in 150 ul mobile phase (100% Methanol with 1mM Ammonium Acetate and 0.2% Formic Acid)