As some acidic phospholipids (mainly phosphatidylserine) are known to complex with calcium, particularly in the presence of Pi, a complete extraction of membrane lipids in mineralized tissues must be done after chemical demineralization (Wu L et al., J Biol Chem 2002, 277, 5126).
Small tissue samples are powdered in the cold (Freezer mill) and extracted with chloroform/methanol (2/1) mixture. After a short sonication (1-2 min), the suspension is centrifuged (3000 rpm, 12 min) and the extract collected. The pellet is demineralized with 0.5M Na salt EDTA for 20 min and sedimented after a short centrifugation. After removal of the supernatant, the decalcified residue (pellet) is reextracted using a chloroform/methanol/conc. HCl (200/100/1) mixture. The two lipid extracts are mixed, dried and washed with saline to remove non-lipid contaminants.