2.2. Fatty Acid Oil Saponification

The fatty acid oil formulation was saponified by the method described by Igarashi and Miyazawa (2000) [16]. The formulations (90 mg each) were incubated in a test tube with nitrogen gas for 15 s. Potassium hydroxide (15 mL; 0.3 N) in 90% methanol was then added and the reaction was incubated at 37 °C for 2 h. The cooled reaction mixture was added to 5 mL of 90% methanol and 15 mL of hexane, and shaken vigorously. Non-saponaceous matter was excluded by washing the methanolic aqueous layer twice with 15 mL of hexane. Hydrochloric acid (9 mL; 6 N) was added to the washed methanolic layer. The fatty acids were obtained by extracting twice with 15 mL of hexane and then evaporating the hexane under a nitrogen stream.