Each sample was next transferred to a 50 mL graduated tube, and 6.25 mL chloroform and 6.25 mL phosphate buffer were added. Each tube was inverted 30 times and the phases allowed to separate for 1 h before recovery of the bottom layer (approximately 12.5 mL). This liquid was transferred to a preweighed aluminum dish and the solvent was evapo- rated over 30 m in a dry oven to 80°C.