The bottom organic layer (chloroform)
was removed and another 10 mL of chloroform
were added to rinse the aqueous phase again (methanolwater),
mixed and centrifuged. The bottom organic layer
was collected and pooled with the first one. The
chloroform was evaporated using a rotary evaporator
(Bu¨ chi, Model R-114, Labortechnik, Switzerland) and
total lipids were dissolved in 15mL of chloroform. Lipid
aliquots (10 mg) from each steak were methylated
separately using acidic (methanolic HCl) and basic
(sodium methoxide) reagents (Kramer et al. 2008). The
fatty acid methyl esters were analyzed using the gas
chromatography (GC) and Ag-HPLC equipment and
methods outlined by Cruz-Hernandez et al.