Sample Extraction
A volume of 25 µL D4-choline (30 µmol/L in water), 0.35 mL water, 1.2 mL methanol and 0.4 mL chloroform were added to 25 µL ample volume and stirred. Afterwards 2.5 µL BHT solution (2.5 mg/mL in ethanol) and 10 µL 1,2-diarachidoyl-sn-glycero-3-phosphocholine (500 µmol/L in trifluoroethanol) was added and the ternary mixture was kept at 4 °C for 1 h. In order to separate the phases, 0.8 mL water and 1.2 mL chloroform were added, then the mixture was vigorously stirred and centrifuged to achieve an upper phase with choline, glycerophosphocholine, phosphocholine and betaine, and a lower phospholipid phase (see above). The upper phase (about 1.6 mL) was adjusted to 2 mL and stored at −80 °C.