Chemostation software

The fatty acid methyl esters (FAMEs) were prepared according to the International Dairy Federation method (IDF 182:2002: ISO 15884:2002, Milkfat: Preparation of fatty acid methyl esters). n-hexane and 2M KOH in methanol were added to the fat sample and the mixture was shaken. Then, the sodium hydrogen sulfate (NaHSO₄) was added, and the mixture was centrifuged (3000 min⁻¹) [66 ]. Chromatographic separation producing FAMEs was carried out using the gas chromatography (GC) technique using a Hewlett-Packard 6890 chromatograph with a flame ionization detector (FID) and a capillary column Supelcowax 10 (length—100 m, inner diameter—0.25 mm, liquid phase—Supelcowax 30, film thickness—0.25 mm; temperature: detector = 250 °C, dispenser = 230 °C, column = 195 °C; carrier gas—helium, flow rate—1.5 mL/min). The identification of fatty acids was performed on the basis of their retention times in relation to the retention times of FAME standards (Supelco 37 Component FAME mixture (10 mg/mL in methylene chloride)). Chemostation software (Agilent, Alpharetta, GA, USA) was used to obtain the percentage of fatty acids, and the amount of fatty acids was expressed as the weight percentage of all FAMEs [67 ].